KR102625189B1 - Combination therapy involving antibodies against claudin 18.2 for treatment of cancer - Google Patents

Abstract

The present invention provides effective treatment and/or prevention of diseases associated with cells expressing CLDN18.2, including cancer diseases such as stomach cancer, esophageal cancer, pancreatic cancer, lung cancer, ovarian cancer, colon cancer, liver cancer, head and neck cancer, and gallbladder cancer and metastatic cancer thereof. Combination therapy is provided for:

Description

Combination therapy using antibodies against Claudin 18.2 in the treatment of cancer {COMBINATION THERAPY INVOLVING ANTIBODIES AGAINST CLAUDIN 18.2 FOR TREATMENT OF CANCER}

Cancer of the stomach and esophagus (GE) is a type of disease with the greatest unmet medical need. Stomach cancer is the second leading cause of death due to cancer worldwide. The incidence of esophageal cancer has been increasing in recent decades, which is consistent with the shift in histological type and primary tumor location. Adenocarcinoma of the esophagus is now more common than squamous cell carcinoma in the United States and the West, with most tumors located in the distal esophagus. Despite established standard treatment methods being aggressive and accompanied by substantial side effects, the overall 5-year survival rate for gastroesophageal cancer is only 20-25%.

The majority of patients have locally advanced or metastatic disease and require first-line chemotherapy. Therapeutics are generally based on a backbone of platinum and fluoropyrimidine derivatives combined with a third compound (such as a taxane or anthracycline). Still, at best, the median progression-free survival can be expected to be 5 to 7 months, and the median overall survival can be expected to be 9 to 11 months.

The lack of significant benefits from the various new generations of combination chemotherapy for these cancers has stimulated research using targeted agents. Recently, Trastuzumab was approved for Her2/neuro-positive gastroesophageal cancer. However, since only ~20% of patients express the target and are suitable for applying this therapy, the medical need remains high.

Claudin 18 splice variant 2 (Claudin 18.2 (CLDN18.2)), a tight junction molecule, is a member of the claudin family of tight junction proteins. CLDN18.2 consists of four membrane-spanning domains, along with two small extracellular loops.

In normal tissues, expression of CLDN18.2 is not detected by RT-PCR, except for the stomach. According to immunohistochemistry using CLDN18.2-specific antibody, the stomach was the only benign tissue.

CLDN18.2 is a highly selective gastric direct antigen expressed only on short-lived differentiated gastric epithelial cells. CLDN18.2 is maintained during the course of malignant transformation and therefore frequently appears on the surface of human gastric cancer cells. Additionally, this pan-tumor antigen is ectopically activated at significant levels in esophageal, pancreatic, and lung adenocarcinomas. CLDN18.2 protein is also localized in lymph node metastases of gastric adenocarcinoma and especially in distant metastases into the ovaries (so-called Krukenberg tumors).

IMAB362, a chimeric IgG1 antibody directed against CLDN18.2, was developed by Ganymed Pharmaceuticals AG. IMAB362 recognizes the first extracellular domain (ECD1) of CLDN18.2 with high affinity and specificity. IMAB362 does not bind to any other claudin family members, including the closely related splice variant 1 of claudin 18 (CLDN18.1). IMAB362 exhibits precise tumor cell specificity and four independent, highly potent mechanisms of action. When IMAB362 binds to its target, it mediates cell death by inducing apoptosis induced by ADCC, CDC, and cross-linking of the target on the tumor cell surface, and by direct inhibition of proliferation. Therefore, IMAB362 effectively lyses CLDN18.2-positive cells, including human gastric cancer cell lines, in vitro and in vivo. Mice producing CLDN18.2-positive cancer cell lines have a survival advantage and treatment with IMAB362 shows tumor remission in up to 40% of mice.

The toxicity and PK/TK profile of IMAB362 were thoroughly evaluated in mice and cynomolgus monkeys, including a dose range finding study in cynomolgus monkeys, a 28-day repeated dose toxicity study, and a 3-month repeated dose toxicity study in mice. carried out. Repeated doses of IMAB362 i.v. were well tolerated in both mice (weekly dosing for a maximum treatment period of 3 months, highest dose level 400 mg/kg) and cynomolgus monkeys (up to 100 mg/kg administered for up to 5 weeks). No signs of systemic or local toxicity were observed. In particular, gastrointestinal toxicity has not been observed in any toxicity studies. IMAB362 does not induce immune activation and cytokine release. No drug effects on male or female reproductive organs were recorded. IMAB362 does not bind to non-target tissues. Biodistribution studies in mice suggest that the lack of gastrointestinal toxicity is probably due to compartmentalization of tight junctions at the luminal site in healthy gastrointestinal epithelium, which appears to severely impede the accessibility of the IMAB362 epitope. . This compartmentalization is lost upon malignant cell transformation, making the epitope druggable by IMAB362.

IMAB362 is in early clinical phase testing. Phase I clinical studies have been conducted in humans. IMAB362 was administered once intravenously to 5 dose cohorts of 3 patients each (33 mg/m ² , 100 mg/m ² , 300 mg/m ² , 600 mg/m ² , 1000 mg/m ² ) and observed for 28 days. did. IMAB362 was very well tolerated in patients with no relevant safety observations. In one patient, all tumor markers measured were significantly reduced within 4 weeks of treatment. IMAB362 is being administered in multiple doses in an ongoing Phase IIa clinical trial.

We present data demonstrating that chemotherapy drugs improve the druggability of CLDN18.2 by anti-CLDN18.2 antibodies such as IMAB362 by stabilizing CLDN18.2 on the surface of cancer cells or increasing its expression. presents. A synergistic effect has been observed between anti-CLDN18.2 antibodies such as IMAB362 and certain chemotherapy regimens, especially those used to treat gastric cancer or human solid tumors. Human cancer cells pretreated by chemotherapy were more susceptible to antibody-induced target-specific killing. In mouse tumor models, tumor control using anti-CLDN18.2 antibody plus chemotherapy is superior to that using anti-CLDN18.2 antibody as a single drug.

Additionally, the data presented herein show that bisphosphonates such as zoledronic acid (ZA), particularly when administered in conjunction with recombinant interleukin-2 (IL-2), inhibit the activity of anti-CLDN18.2 antibodies such as IMAB362. It indicates an increase. The underlying mechanism is the activation and expansion of a highly cytotoxic immune cell population (γ9δ2 T cells).

Summary of the invention

The present invention generally relates to gastric cancer, esophageal cancer, pancreatic cancer, lung cancer such as non-small cell lung cancer (NSCLC), ovarian cancer, colon cancer, liver cancer, head and neck cancer, and gallbladder cancer and their metastases, especially gastric cancer metastases such as Krukenberg tumors, abdominal cancer, Combination therapies are provided to effectively treat and/or prevent diseases associated with cells expressing CLDN18.2, including cancer diseases such as metastases and lymph node metastases. Particularly preferred cancer diseases are adenocarcinomas of the stomach, esophagus, pancreatic duct, biliary tract, lung and ovary.

In one aspect, the present invention provides a method for treating or preventing a cancer disease, comprising administering to a patient an antibody that has the ability to bind CLDN18.2 in combination with an agent that stabilizes or increases the expression of CLDN18.2. provides. Expression of CLDN18.2 preferably occurs on the cell surface of cancer cells. An agent that stabilizes or increases the expression of CLDN18.2 may be added before, simultaneously with, or after administration of an antibody capable of binding to CLDN18.2 or a combination thereof.

Agents that stabilize or increase expression of CLDN18.2 may be cytotoxic and/or cytostatic. In one embodiment, the agent that stabilizes or increases the expression of CLDN18.2 causes cell cycle arrest or cell accumulation in one or more phases of the cell cycle, preferably in one or more phases of the cell cycle other than the G1-phase. Contains inducing agents. Agents that stabilize or increase expression of CLDN18.2 may include agents selected from the group consisting of anthracyclines, platinum compounds, nucleoside analogs, taxanes, and camptothecin analogs, or prodrugs thereof, and combinations thereof. . Agents that stabilize or increase expression of CLDN18.2 may include agents selected from the group consisting of epirubicin, oxaliplatin, cisplatin, 5-fluorouracil or prodrugs thereof such as capecitabine, docetaxel, irinotecan, and combinations thereof. You can. Agents that stabilize or increase the expression of CLDN18.2 include a combination of oxaliplatin and 5-fluorouracil or its prodrug, a combination of cisplatin and 5-fluorouracil or a prodrug thereof, and one or more anthracyclines with oxaliplatin. Combination, combination of one or more anthracyclines with cisplatin, combination of one or more anthracyclines with 5-fluorouracil or its prodrug, combination of one or more taxanes with oxaliplatin, combination of one or more taxanes with cisplatin, one or more It may include a combination of a taxane with 5-fluorouracil or a prodrug thereof, or a combination of one or more camptothecin analogs with 5-fluorouracil or a prodrug thereof. Agents that stabilize or increase expression of CLDN18.2 may be agents that induce immunogenic cell death. Agents that induce immunogenic cell death may include agents selected from the group consisting of anthracyclines, oxaliplatin, and combinations thereof. Agents that stabilize or increase expression of CLDN18.2 may include a combination of epirubicin and oxaliplatin. In one embodiment, the method of the invention comprises administering at least one anthracycline, at least one platinum compound, and at least one 5-fluorouracil and its prodrugs. The anthracycline may be selected from the group consisting of epirubicin, doxorubicin, daunorubicin, idarubicin, and valrubicin. Preferably, the anthracycline is epirubicin. The platinum compound may be selected from the group consisting of oxaliplatin and cisplatin. The nucleoside analog may be selected from the group consisting of 5-fluorouracil and its prodrugs. The taxane may be selected from the group consisting of docetaxel and paclitaxel. Camptothecin analogs may be selected from the group consisting of irinotecan and topotecan. In one embodiment, the method of the invention comprises (i) epirubicin, oxaliplatin, and 5-fluorouracil, (ii) epirubicin, oxaliplatin, and capecitabine, (iii) epirubicin, cisplatin, and 5-fluorouracil, louracil, (iv) epirubicin, cisplatin and capecitabine, or (v) folinic acid, oxaliplatin and 5-fluorouracil.

In one embodiment, the method of the invention further comprises administering an agent that stimulates γδ T cells. In one embodiment, the γδ T cells are Vγ9Vδ T cells. In one embodiment, the agent that stimulates γδ T cells is a bisphosphonate, such as a nitrogen-containing bisphosphonate (aminophosphonate). In one embodiment, the agent that stimulates γδ T cells is zoledronic acid, clodronic acid, ibandronic acid, pamidronic acid, risedronic acid, minodronic acid, olpadronic acid, alendronic acid, incadronic acid, and salts thereof. is selected from the group consisting of In one embodiment, an agent that stimulates γδ T cells is administered in combination with interleukin-2.

The method of the invention may further comprise administering at least one additional chemotherapy agent, which may be a cytotoxic agent.

Antibodies capable of binding to CLDN18.2 can bind to the natural epitope of CLDN18.2 present on the surface of living cells. In one embodiment, the antibody capable of binding to CLDN18.2 binds to the first extracellular loop of CLDN18.2. In one embodiment, the antibody having the ability to bind CLDN18.2 is an antibody that binds cells by one or more of complement-dependent cytotoxicity (CDC)-mediated lysis, antibody-dependent cytotoxicity (ADCC)-mediated lysis, induction of apoptosis, and inhibition of proliferation. Mediates death. In one embodiment, the antibody capable of binding CLDN18.2 is a monoclonal, chimeric or humanized antibody, or antibody fragment. In one embodiment, the antibody having the ability to bind CLDN18.2 is (i) accession numbers DSM ACC2737, DSM ACC2738, DSM ACC2739, DSM ACC2740, DSM ACC2741, DSM ACC2742, DSM ACC2743, DSM ACC2745, DSM ACC2746, DSM ACC2747 , an antibody produced and/or obtainable from a clone deposited as DSM ACC2748, DSM ACC2808, DSM ACC2809, or DSM ACC2810, (ii) an antibody that is a chimeric or humanized form of the antibody of (i) above, (iii) an antibody of (i) above ) and (iv) preferably an antibody having the specificity of the antibody of (i) above and comprising an antigen-binding portion or antigen-binding site, especially a variable region, of the antibody of (i). It is an antibody. In one embodiment, the antibody is coupled to a therapeutic agent such as a toxin, radioisotope, drug, or cytotoxic agent.

In one embodiment, the method of the invention comprises administering an antibody capable of binding to CLDN18.2 at a dosage of up to 1000 mg/m ² . In one embodiment, the method of the invention comprises repeated administration of an antibody capable of binding to CLDN18.2 at a dosage of 300 to 600 mg/m ² .

In one embodiment, the cancer is CLDN18.2 positive. In one embodiment, the cancer disease is selected from the group consisting of stomach cancer, esophageal cancer, pancreatic cancer, lung cancer, ovarian cancer, colon cancer, liver cancer, head and neck cancer, gallbladder cancer, and metastases thereof. The cancerous condition may be a Krukenberg tumor, peritoneal metastases, and/or lymph node metastases. In one embodiment, the cancer is adenocarcinoma, particularly advanced adenocarcinoma. In one embodiment, the cancer is selected from the group consisting of cancer of the stomach, cancer of the esophagus, especially cancer of the lower esophagus, cancer of the gastroesophageal junction, and gastroesophageal cancer. Patients may be HER2/neu negative or HER2/neu positive but not treatable with trastuzumab therapy.

According to the present invention, CLDN18.2 preferably has an amino acid sequence according to SEQ ID NO: 1.

In another aspect, the present invention provides a medical preparation comprising an antibody capable of binding to CLDN18.2 and an agent that stabilizes or increases the expression of CLDN18.2. The pharmaceutical preparation of the present invention may further include an agent that stimulates γδ T cells. The antibody capable of binding to CLDN18.2 and the agent that stabilizes or increases the expression of CLDN18.2 and, optionally, the agent that stimulates γδ T cells may be present in the pharmaceutical preparation as a mixture or separately from each other. The pharmaceutical preparation comprises a first container comprising an antibody capable of binding to CLDN18.2 and a container comprising an agent that stabilizes or increases the expression of CLDN18.2, and optionally an agent that stimulates γδ T cells. It may be a kit containing: The pharmaceutical preparation may also further comprise printed instructions regarding the use of the agent for treating cancer, particularly the agent used in the method of the present invention. Several different embodiments of the pharmaceutical preparation, particularly agents that stabilize or increase the expression of CLDN18.2 and agents that stimulate γδ T cells, are as described above in connection with the method of the invention.

The invention also provides an agent that has the ability to bind CLDN18.2 for use in the methods described herein, such as for administration in combination with an agent that stabilizes or increases the expression of CLDN18.2 and optionally an agent that stimulates γδ T cells. Agents such as antibodies are also provided.

Other features and advantages of the present invention will become apparent from the following detailed description and claims.

Figure 1. Effect of chemotherapy on gastric cancer cells. KatoIII cells were cultured for 96 hours to arrest the cell cycle in G0/G1-phase and induce downregulation of CLDN18.2. Cytostatic Compounds that result in cell cycle arrest in various cell cycle phases (S-phase (5-FU) or G2-phase (epirubicin)) stabilize CLDN18.2-expression.
Figure 2. Effect of chemotherapy on gastric cancer cells. a/b: Effect of chemotherapy on transcript and protein levels of CLDN18.2 in gastric cancer cells. c: Flow cytometry analysis of extracellular IMAB362 binding to gastric cancer cells treated with chemotherapy agents.
Figure 3. Effect of chemotherapy on gastric cancer cells. Cytostatic compounds that arrest the cell cycle in various phases of the cell cycle (S/G2-phase (irinotecan) or G2-phase (docetaxel)).
Figure 4. IMAB362-induced ADCC directed killing of gastric cancer cells after chemotherapy drug pretreatment.
Figure 5. Effect of chemotherapy on gastric cancer cells. a: irinotecan, Cells treated with docetaxel or cisplatin showed lower levels of viable cells compared to medium-cultured target cells. b: CLDN18.2 expression in cells treated with irinotecan, docetaxel or cisplatin is increased compared to medium cultured cells. c/d: Treatment of cells with irinotecan, docetaxel or cisplatin enhances the ability of IMAB362 to induce ADCC.
Figure 6. Effect of chemotherapy on IMAB362-induced CDC.
Figure 7. Effect of chemotherapy on effector cells.
Figure 8. Expansion of PBMCs in ZA/IL-2 supplemented cultures.
Figure 9. Enrichment of Vγ9Vδ2 T cells in ZA/IL-2 supplemented PBMC cultures.
Figure 10. Enrichment of Vγ9Vδ2 T cells and increased IL-2 dose in medium supplemented with ZA.
Figure 11. Expansion and cytotoxic activity of Vγ9Vδ2 T cells after incubation with ZA-pulsed monocytes and human cancer cells.
Figure 12. ZA-dependent development of various cell types in PBMC cultures.
Figure 13. Display of surface markers on Vγ9Vδ2 T-cells after ZA/IL-2 treatment.
Figure 14. ADCC activity of Vγ9Vδ2 T cells by IMAB362 against CLDN18.2-positive NUGC-4 gastric cancer cells.
Figure 15. ADCC of IMAB362 using Vγ9Vδ2 T cells as effector cells.
Figure 16. Effect of ZA on surface localization of CLDN18.2 on target cells.
Figure 17. Effect of ZA/IL-2 treatment and chemotherapy on effector cells.
Figure 18. Biodistribution study using conjugated antibodies in mice
Figure 19. Early treatment of HEK293~CLDN18.2 tumor xenografts.
Figure 20. Treatment of advanced HEK293~CLDN18.2 tumor xenografts.
Figure 21. Effect of IMAB362 on subcutaneous tumor growth of gastric cancer xenografts.
Figure 22. Effect of immunotherapy using IMAB362 on NCI-N87~CLDN18.2 gastric carcinoma xenografts
Figure 23. Effect of combined treatment of IMAB362 and EOF on NCI-N87~CLDN18.2 xenografts.
Figure 24. Effect of IMAB362 and EOF combination treatment on NUGC-4~CLDN18.2 xenografts.
Figure 25. Effect of ZA/IL-2 induced Vγ9Vδ2 T cells on control of macroscopic tumors by IMAB362 in NSG mice.
Figure 26. Combination treatment effect of IMAB362 and EOF on CLS-103~cldn18.2 allograft tumor.

Although the invention is described in more detail below, it should be understood that the invention is not limited to the specific methodologies, protocols and reagents described herein. Additionally, the terms used in this specification are only for describing specific embodiments, and should not be construed as intended to limit the scope of the present invention, and the scope of the present invention should be understood to be limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

In the following, elements of the present invention are described. Although these components are listed with specific embodiments, it should be understood that they can be combined in any way and any number of times to create additional embodiments. The various embodiments and preferred embodiments described herein should not be construed as limiting the invention to only the explicitly described embodiments. This description is to be understood as supporting and encompassing embodiments that combine any number of the disclosed and/or preferred elements with the explicitly described embodiments. Moreover, any permutations and combinations of all described elements of this application are to be considered disclosed by the description of this application unless the context otherwise dictates.

Preferably, the terms used herein are those defined in “A multilingual glossary of biotechnological terms: (IUPAC Recommendations)”, H.G.W. Defined as described in Leuenberger, B. Nagel, and H. Koebl, Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

In practicing the present invention, unless otherwise stated, the literature in the art (cf., e.g., Molecular Cloning: A Laboratory Manual , ^2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989), methods of chemistry, biochemistry, cell biology, immunology, and recombinant DNA technology will be used.

Throughout the following detailed description and claims, unless the context otherwise requires, "comprise" and its variants such as "comprises" and "comprising" refer to the specified member, integer or step or to the member, integer or step. encompasses groups of elements, but does not exclude other members, integers or stages or groups of members, integers or stages, although in some embodiments other members, integers or stages or groups of members, integers or stages may be excluded. That is, the claimed subject matter of the present invention includes the specified member, integer or step or groups of members, integer or step. In describing the invention, the articles "a", "an" and "the" and similar reference terms used in the context (particularly in the context of the claims) unless otherwise stated or clear to have the opposite meaning, It encompasses both singular and plural.

Recitation of values herein as ranges is merely for the convenience of individually referring to each individual value within that range. Unless otherwise indicated, each individual value is deemed to be incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any appropriate order unless otherwise indicated or otherwise contradicted by context. Any and all example or exemplary language (e.g., “such as”) provided herein is intended to better explain the invention and is not intended to otherwise limit the scope of the invention. No language in the specification should be construed as indicating any non-claimed element as essential to practicing the invention.

Several documents are cited throughout the specification of the present invention. Each of the above or below cited documents (all patents, patent applications, scientific publications, manufacturer's instructions, instructions, etc.) is hereby incorporated by reference in its entirety. None of these cited documents should be construed as an admission that the present invention is not said to antedate such disclosure simply because it is a prior invention.

The term “CLDN18” refers to Claudin 18 and encompasses any variants, including Claudin 18 splice variant 1 (Claudin 18.1 (CLDN18.1)) and Claudin 18 splice variant 2 (Claudin 18.2 (CLDN18.2)). .

The term "CLDN18.2" preferably relates to human CLDN18.2, preferably comprising an amino acid sequence according to SEQ ID NO: 1 of the sequence listing or a variant of said amino acid sequence, preferably to such a sequence. It relates to the proteins it consists of.

The term "CLDN18.1" preferably relates to human CLDN18.1 and in particular to a protein comprising, preferably consisting of, an amino acid sequence according to SEQ ID NO: 2 of the sequence listing or a variant of said amino acid sequence. It's about.

In the present invention, the term "variant" particularly refers to mutants, splice variants, conformations, isoforms, allelic variants, species variants, and species homologs ( species homologs), especially those that exist naturally. Allelic variants involve alterations in the normal sequence of a gene, the significance of which is often uncertain. Complete genetic sequencing for a given gene often identifies numerous allelic variants. A species homolog is a nucleic acid or amino acid sequence from another species that differs in origin from that of a given nucleic acid or amino acid sequence. The term “variant” encompasses both post-translationally modified and conformational variants.

According to the present invention, the term “CLDN18.2 positive cancer” means a cancer comprising cancer cells expressing CLDN18.2, preferably cancer cells expressing CLDN18.2 on the surface of said cancer cells.

The term “cell surface” is used in its usual sense in the art and thus includes the exterior of the cell accessible to proteins and other molecules.

CLDN18.2 is expressed on the cell surface if it is located on the surface of the cell and is accessible for binding by CLDN18.2-specific antibodies added to the cell.

In the present invention, CLDN18.2 is not actually expressed in cells if the expression level is lower than that in gastrointestinal cells or gastrointestinal tissue. Preferably, the expression level is less than 10%, preferably less than 5%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.1% or 0.05% of the expression level in gastric cells or gastrointestinal tissue. Less than or even lower is preferred. Preferably, CLDN18.2 is not substantially expressed in cells if the expression level exceeds the expression level in non-cancerous tissues other than the stomach by 2-fold or less, preferably 1.5-fold or less. Ideally, it is better not to exceed the expression level in non-cancerous tissues. Preferably, CLDN18.2 is not actually expressed in the cell if the expression level is below the limit of detection and/or is too low to allow binding by CLDN18.2-specific antibodies added to the cells.

According to the present invention, CLDN18.2 has an expression level that preferably exceeds the expression level in non-cancerous tissues other than the stomach by a factor of 2, preferably by a factor of 10, greater than 100, greater than 1000, or greater than 10000-fold. Expressed in cells. Preferably, CLDN18.2 is expressed in the cell when the level of expression is above the limit of detection and/or is high enough to allow binding by a CLDN18.2-specific antibody added to the cell. Preferably, the CLDN18.2 expressed in the cell is expressed or exposed on the surface of the cell.

According to the present invention, the term “disease/condition” refers to any pathological condition, including cancer, especially the types of cancer described herein. Any designation for cancer or a specific form of cancer also encompasses its cancerous metastases. In a preferred embodiment, the disease to be treated according to the present application is associated with cells expressing CLDN18.2.

“Disease associated with cells expressing CLDN18.2” or similar expressions mean, according to the invention, that CLDN18.2 is expressed in cells of the diseased tissue or organ. In one embodiment, the expression of CLDN18.2 in cells of a diseased tissue or organ is increased compared to the state in a healthy tissue or organ. An increase means an increase of at least 10%, especially at least 20%, at least 50%, at least 100%, at least 200%, at least 500%, at least 1000%, at least 10000% or more. In one embodiment, expression is found only in diseased tissue, whereas expression is suppressed in healthy tissue. According to the present invention, diseases associated with cells expressing CLDN18.2 include cancer diseases. Additionally, according to the present invention, the cancer disease is preferably a disease in which cancer cells express CLDN18.2.

In the present invention, “cancer disease” or “cancer” encompasses diseases characterized by abnormal regulation of cell growth, proliferation, differentiation, adhesion and/or migration. “Cancer cell” means an abnormal cell that grows by rapid, uncontrolled cell proliferation and continues to grow even after the stimulus that initiated the new growth has ceased. Preferably, the “cancer disease” is characterized by cells expressing CLDN18.2 and the cancer cells express CLDN18.2. The cells expressing CLDN18.2 are preferably cancer cells, preferably cancer cells of the cancer described in the present invention.

“Adenocarcinoma” is a cancer that originates in glandular tissue. This tissue is also part of a larger tissue category known as epithelial tissue. Epithelial tissue includes skin, glands, and various other tissues that line the body's cavities and organs. Epithelium is embryologically derived from ectoderm, endoderm and mesoderm. As long as the cells have secretory properties, they do not necessarily have to be part of a gland to be classified as an adenocarcinoma. This form of carcinoma can occur in some higher mammals, including humans. Well-differentiated adenocarcinomas tend to resemble the glandular tissue from which they originate, whereas poorly differentiated adenocarcinomas may not. By staining the cells in a biopsy, a pathologist can determine whether the tumor is adenocarcinoma or another type of cancer. Adenocarcinoma can occur in many tissues of the body due to the fact that glands exist throughout the body. Each gland may not secrete the same agent, but as long as it has an exocrine function for the cells, it is considered a gland and its malignant form is therefore named adenocarcinoma. Malignant adenocarcinoma invades other tissues and often metastasizes if given enough time. Ovarian adenocarcinoma is the most common type of ovarian carcinoma. This includes serous and mucinous adenocarcinoma, clear cell adenocarcinoma, and endometrioid adenocarcinoma.

“Metastasis” means the spread of cancer cells from their original site to other parts of the body. The formation of metastases is a very complex process and depends on the detachment of malignant cells from the tumor, invasion of the extracellular matrix, entry into body cavities and blood vessels by invasion of the epithelial basement membrane, and subsequent invasion into target organs after transport by the blood. Finally, new tumor growth at the target site depends on angiogenesis. Tumor metastases often occur because tumor cells or components may remain or develop metastatic potential even after removal of the primary tumor. In one embodiment, the term “metastasis” according to the present invention relates to “distant metastasis”, which refers to metastasis distant from the primary tumor and regional lymph node system. In one embodiment, the term “metastasis” according to the present invention relates to lymph node metastasis. One particular type of metastasis treatable with the treatment of the present invention is metastasis originating from gastric cancer as the primary site. In a preferred embodiment, such gastric cancer metastases are Krukenberg tumors, peritoneal metastases and/or lymph node metastases.

Krukenberg tumor is an uncommon metastatic tumor of the ovary, accounting for 1% to 2% of all ovarian tumors. The prognosis for Krukenberg tumor remains poor, and there is no established treatment for Krukenberg tumor. Krukenberg tumor is a metastatic signet cell adenocarcinoma of the ovary. The stomach is the primary site of most Krukenberg tumor cases (70%). Carcinomas of the colon, appendix, and breast (mainly invasive lobular carcinoma) are the second most common primary sites. Uncommon cases of Krukenberg tumors originating from carcinomas of the gallbladder, biliary tract, pancreas, small intestine, ampulla of Vater, cervix, and bladder/urachus have also been reported. The interval between diagnosis of primary carcinoma and subsequent discovery of ovarian involvement is usually less than 6 months, but longer periods have been reported. In many cases, the primary tumor is so small that it is often missed during examination. A previous history of carcinoma of the stomach or other organs occurs in only 20% to 30% of cases.

Krukenberg tumor is the most common example of selective spread of cancer in the gastro-ovarian axis. This axis of tumor spread has historically attracted the attention of many pathologists, especially when gastric neoplasms spread to other tissues. This was especially true if it was found to have spread selectively to the ovaries without involvement. The route of metastasis of gastric carcinoma to the ovary has long been a mystery, but it is now clear that retrograde lymphatic spread is the most likely route of this metastasis.

Women with Krukenberg tumor tend to be unusually young, with an average age of 45 years, compared to patients with metastatic carcinoma, who are typically in their 50s. This young age distribution may be related, in part, to the increased incidence of gastrointestinal signet signet cell carcinoma in young women. Common symptoms are usually related to the ovaries, the most common being abdominal pain and bloating (often due to large bilateral ovarian masses). The remaining patients present with non-specific gastrointestinal symptoms or are asymptomatic. Additionally, Krukenberg tumor has been reported to be associated with virilization due to hormone production by the ovarian stroma. In 50% of cases of this disease, ascites are present, usually manifesting as malignant cells.

Krukenberg tumors are bilateral in >80% of reported cases. The ovaries usually grow asymmetrically and have a bosselated outline. The compartmentalized surface is yellow or white; They are usually solid, although they are often cystic. It is important to note that the capsular surface of the ovary containing a Krukenberg tumor is usually smooth and free of adhesions or peritoneal deposits. This is interesting given that other metastatic tumors to the ovary have been associated with superficial implants. This may explain why the gross morphology of Krukenberg tumors may appear deceptively like primary ovarian tumors. However, the bilateral nature of Krukenberg tumor remains consistent in its metastatic characteristics.

Patients with Krukenberg tumors have a significantly higher overall mortality rate. Most patients die within 2 years (median survival rate, 14 months). Several studies have shown that the prognosis is poor if the primary tumor is identified after metastasis to the ovaries has been discovered, and that the prognosis is worse if the primary tumor remains hidden.

In the literature, the optimal treatment strategy for Krukenberg tumor has not been clearly established so far. There are also no adequate guidelines as to whether surgical resection should be performed. Chemotherapy or radiotherapy has no significant effect on the prognosis of patients with Krukenberg tumor.

“Treat” means reducing the size or number of tumors in a subject; Stopping or slowing the progression of a subject's disease; Inhibiting or slowing the development of new diseases in a subject; Reducing the frequency or severity of symptoms and/or relapses in subjects who currently have the disease or have previously had the disease; and/or administering a compound or composition or combination of compounds or compositions to a subject in order to prevent or eliminate a disease, including prolonging, i.e. increasing, the lifespan of the subject.

In particular, the term "treatment of a disease" includes curing, shortening, alleviating, preventing, slowing, inhibiting the progression or worsening, or preventing or slowing the onset of the disease or its symptoms.

As used herein, the term "patient" refers to a subject of treatment, especially a subject suffering from a disease, including humans, non-human primates or other animals, especially mammals such as cattle, horses, pigs, sheep, goats, dogs, cats, or This includes rodents such as mice and rats. In a particularly preferred embodiment, the patient is a human.

The term “an agent that stabilizes or increases the expression of CLDN18.2” refers to the effect of CLDN18.2 when a cell is provided with the agent or combination of agents compared to the situation in a cell not provided with such agent or combination of agents. refers to an agent or combination of agents that results in increased RNA and/or protein levels of 2, preferably resulting in increased levels of CLDN18.2 protein on the surface of the cell. Preferably, the cells are cancer cells, especially cells expressing CLDN18.2, such as cells of the types of cancer described herein. The term "an agent that stabilizes or increases the expression of CLDN18.2" refers specifically to the effect that, when a cell is provided with the agent or combination of agents, compared to the situation in a cell that is not provided with such agent or combination of agents. Refers to an agent or combination of agents that results in a higher density of CLDN18.2 on the surface of cells. The expression “stabilize the expression of CLDN18.2” specifically includes situations where the agent or combination of agents prevents or reduces the decrease in expression of CLDN18.2, e.g. the agent or combination of agents This applies to cases in which the expression of CLDN18.2 is reduced if not provided, or the reduction in CLDN18.2 expression is prevented or the reduction in expression is reduced by providing the agonist or a combination of agonists. The expression “increase the expression of CLDN18.2” particularly includes situations where the agent or combination of agents increases the expression of CLDN18.2, e.g. when the agent or combination of agents is not provided. In situations where the expression of CLDN18.2 is essentially reduced or maintained constant and when an agent or combination of agents is provided, compared to the situation in cells not provided with such agent or combination of agents, the expression of CLDN18.2 By increasing expression, this may result in a situation where CLDN18.2 is expressed at a higher level compared to a situation where the expression of CLDN18.2 is basically lowered or maintained constant because such agent or combination of agents is not provided. Included.

According to the present invention, the term “agent that stabilizes or increases the expression of CLDN18.2” includes chemotherapeutic agents or combinations of chemotherapeutic agents, such as cytostatic agents. Chemotherapeutic agents can either: (1) damage a cell's DNA, preventing it from proliferating, or (2) inhibit the synthesis of new DNA strands, making the cell unable to replicate. (3) It can affect cells by stopping the cell's mitotic process, preventing the cell from dividing into two cells.

According to the present invention, the term "agent that stabilizes or increases the expression of CLDN18.2" preferably refers to an agent that, when given to cells, particularly cancer cells, induces cell cycle arrest or arrest of one or more phases of the cell cycle. , preferably a cell cycle other than the G1- and G0-phases, preferably a cell cycle other than the G1-phase, preferably one or more of the G2- or S-phases of the cell cycle, such as G1/ of the cell cycle. Agents or combinations of agents, such as cytostatic compounds or combinations of cytostatic compounds, which result in arrest of cells or accumulation of cells in G2-, S/G2-, G2- or S-phase. all. The expression “cells arrest or accumulate in one or more phases of the cell cycle” means that the percentage of cells in said one or more phases of the cell cycle increases. Each cell must go through the four phases of the cell cycle to replicate itself. The first phase, called G1, is when the cell is ready to replicate its own dye. The second stage is called S. In this phase, DNA synthesis occurs and DNA is replicated. The next phase is the G2 phase, when RNA and proteins are replicated. The last phase is the M phase, where cells actually divide. In this final step, the replicated DNA and RNA divide and travel to the separate ends of the cell, effectively dividing the cell into two identical, functional cells. Chemotherapeutic agents, which are DNA-damaging agents, usually result in cells accumulating in G1 and/or G2 phase. Chemotherapeutic agents that block cell growth by interfering with DNA synthesis, such as antimetabolites, usually result in cells accumulating in S-phase. Examples of these drugs include 6-mercaptopurine and 5-fluorouracil.

According to the present invention, the term "agent that stabilizes or increases the expression of CLDN18.2" includes anthracyclines such as epirubicin, platinum compounds such as oxaliplatin and cisplatin, nucleosides such as 5-fluorouracil or prodrugs thereof. analogs, taxanes such as docetaxel, and camptothecin analogs such as irinotecan and topotecan, and combinations of drugs, such as anthracyclines such as epirubicin, oxaliplatin and one or more of 5-fluorouracil, such as oxaliplatin and combinations of drugs containing 5-fluorouracil or other drug combinations described herein.

In one preferred embodiment, the “agent that stabilizes or increases the expression of CLDN18.2” is an “agent that induces immunogenic cell death.”

Under certain circumstances, cancer cells can enter a lethal stress pathway linked to the release of a combination of spatiotemporally defined signals that are decoded by the immune system and activate a tumor-specific immune response (Zitvogel L. et al. (2010) Cells 140: 798-804). Under this scenario, cancer cells are triggered to release signals that are sensed by innate immune effectors such as dendritic cells, triggering a cognate immune response involving CD8+ T cells and IFN-γ signaling, resulting in tumor cell death. It can elicit a productive anti-cancer immune response. These signals include pre-apoptotic exposure of the endoplasmic reticulum (ER) chaperone calreticulin (CRT) to the cell surface, pre-apoptotic secretion of ATP, and post-apoptotic release of the nuclear protein HMGB1. Together, these processes constitute the molecular determinants of immunogenic cell death (ICD). Anthracyclines, oxaliplatin, and γ irradiation can induce all the signals that define ICD, whereas cisplatin lacks the induction of CRT translocation from the ER to the surface of dying cells - a process that requires ER stress. Such agonists require complementation by the ER stress inducer thapsigargin.

According to the present invention, the term "agent that induces immunogenic cell death" refers to an agent that, when provided to cells, particularly cancer cells, can cause those cells to enter a lethal stress pathway that ultimately results in a tumor-specific immune response. Refers to an agent or combination of agents. In particular, agents that induce immunogenic cell death, when provided to cells, cause the cells to undergo pre-apoptotic exposure of the endoplasmic reticulum (ER) chaperone calreticulin (CRT) on the cell surface and pre-apoptotic secretion of ATP. and post-apoptotic release of the nuclear protein HMGB1.

In the present invention, the term “agent that induces immunogenic cell death” includes anthracyclines and oxaliplatin.

Anthracyclines are also antibiotic agents and are a class of drugs commonly used in cancer chemotherapy. Structurally, all anthracyclines share a common tetracyclic 7,8,9,10-tetrahydrotetracene-5,12-quinone structure and usually require glycosylation at specific sites.

Anthracyclines preferably have one or more of the following actions: 1. Prevent replication of rapidly growing cancer cells by inhibiting DNA and RNA synthesis by intercalating between base pairs of DNA/RNA strands. 2. Blocks DNA transcription and replication by inhibiting the topoisomerase II enzyme, preventing relaxation of supercoiled DNA. 3. Generates iron-mediated free oxygen radicals that damage DNA and cell membranes.

According to the present invention, “anthracycline” refers to an agent that induces apoptosis, preferably by inhibiting the recombination of DNA in topoisomerase II, preferably an anticancer agent.

Preferably, in the present invention, “anthracycline” generally refers to compounds having the following ring structures, their analogs and derivatives, pharmaceutical salts, hydrates, esters, conjugates and prodrugs.

Examples of anthracyclines and anthracycline analogs include daunorubicin (daunomycin), doxorubicin (Adriamycin), epirubicin, idarubicin, rhodamycin, pyrarubicin, valrubicin, and N-trifluoro-acetyl doxorubicin. -14-valerate, aclacinomycin, morpholinodoxorubicin (Morpholino-DOX), cyanomorpholino-doxorubicin (Cyanomorpholino-DOX), 2-pyrrolino-doxorubicin (2- PDOX), 5-iminodaunomycin, mitoxantrone, and aclacinomycin A (aclarubicin), but are not limited thereto. Mitoxantrones are a member of the anthracenedione class of compounds, which are anthracycline analogues that lack the sugar moieties of anthracyclines but retain the planar polycyclic aromatic ring structure that allows them to intercalate into DNA.

Particularly preferred as anthracyclines according to the invention are compounds having the following structure:

During the ceremony

R ₁ is selected from the group consisting of H and OH, R ₂ is selected from the group consisting of H and OMe, R ₃ is selected from the group consisting of H and OH, R ₄ is selected from the group consisting of H and OH do.

In one embodiment, R ₁ is H, R ₂ is OMe, R ₃ is H and R ₄ is OH. In another embodiment, R ₁ is OH, R ₂ is OMe, R ₃ is H and R ₄ is OH. In another embodiment, R ₁ is OH, R ₂ is OMe, R ₃ is OH and R ₄ is H. In another embodiment, R ₁ is H, R ₂ is H, R ₃ is H, and R ₄ is OH.

Considered to be particularly preferred as anthracycline in the context of the present invention is epirubicin. Epirubicin is an anthracycline drug with the following structure:

It is sold under the brand name Ellence in the United States and under the brand names Pharmorubicin or Epirubicin Ebewe in other countries. In particular, the term "epirubicin" refers to the compound (8R,10S)-10-[(2S,4S,5R,6S)-4-amino-5-hydroxy-6-methyl-oxan-2-yl]oxy -6,11-dihydroxy-8-(2-hydroxyacetyl)-1-methoxy-8-methyl-9,10-dihydro-7H-tetracene-5,12-dione. Epirubicin is preferred over doxorubicin, the most widely used anthracycline, in some chemotherapy regimens because epirubicin has been shown to have fewer side effects.

According to the present invention, the term “platinum compound” refers to a compound containing platinum in its structure, such as a platinum complex, and includes compounds such as cisplatin, carboplatin and oxaliplatin.

The term “cisplatin” or “cisplatinum” refers to cis -diaminedichloroplatinum(II) (CDDP), which has the following structure:

“Carboplatin” refers to the cis-diamine(1,1-cyclobutanedicarboxylate)platinum(II) compound having the following structure:

The term “oxaliplatin” refers to a compound that is a platinum compound complexed to a diaminocyclohexane carrier ligand having the following structural formula:

In particular, the term "oxaliplatin" refers to the compound [(1R,2R)-cyclohexane-1,2-diamine](ethanedioato-O,O')platinum(II) compound. Injectable oxaliplatin is also sold under the brand name Eloxatine.

“Nucleoside analog” refers to a structural analog of a nucleoside, and this category includes both purine analogs and pyrimidine analogs. In particular, “nucleoside analog” refers to fluoropyrimidine derivatives, including fluorouracil and its prodrugs.

“Fluorouracil” or “5-fluorouracil” (5-FU or f5U) (sold under the brand names Adrucil, Carac, Efudix, Efudex and Fluoroplex) is a compound that is a pyrimidine analog with the structure:

In particular, the term refers to the compound 5-fluoro-1H-pyrimidine-2,4-dione.

“Capecitabine” (Xeloda, Roche) refers to a chemotherapy agent that is a prodrug that is converted to 5-FU in tissues. Orally administrable capecitabine has the following structural formula:

In particular, this term refers to the compound pentyl [1-(3,4-dihydroxy-5-methyltetrahydrofuran-2-yl)-5-fluoro-2-oxo-1H-pyrimidin-4-yl]carba Point to mate.

Taxanes are a class of diterpene compounds that were initially derived from natural sources such as plants of the genus Taxus, but some types have been artificially synthesized. The main mechanism of action of the taxane class of compounds is by disruption of microtubule function, thereby inhibiting the cell division process. Taxanes include docetaxel (Taxotere) and paclitaxel (Taxol).

In the present invention, “docetaxel” refers to a compound with the following structure:

In the present invention, “paclitaxel” refers to a compound with the following structure:

In the present invention, the term "camptothecin analog" refers to camptothecin (CPT; (S)-4-ethyl-4-hydroxy-1H-pyrano[3',4':6,7]indolizino[1 ,2-b] refers to a derivative of the quinoline-3,14-(4H,12H)-dione) compound. Preferably, the “camptothecin analog” is a compound having the following structure:

In the present invention, preferred camptothecin analogs are inhibitors of the DNA enzyme topoisomerase I (topo I). Preferred camptothecin analogs according to the invention are irinotecan and topotecan.

Irinotecan is a drug that prevents DNA from unwinding by inhibiting topoisomerase I. In chemical terms, it is a semisynthetic analogue of the natural alcoholoid camptothecin with the following structure:

In particular, the term "irinotecan" refers to the compound (S)-4,11-diethyl-3,4,12,14-tetrahydro-4-hydroxy-3,14-dioxo1H-pyrano[3',4 ':6,7]-indolizino[1,2-b]quinolin-9-yl-[1,4'bipiperidine]-1'-carboxylate.

Topotecan is a topoisomerase inhibitor with the following structure:

In particular, the term "topotecan" refers to the compound (S)-10-[(dimethylamino)methyl]-4-ethyl-4,9-dihydroxy-1H-pyrano[3',4':6,7] Refers to indolizino[1,2-b]quinoline-3,14(4H,12H)-dione monohydrochloride.

According to the present invention, the agent that stabilizes or increases the expression of CLDN18.2 may be a chemotherapy agent, in particular a chemotherapy agent established for use in the treatment of cancer and may be a combination of drugs such as combinations of drugs established for use in the treatment of cancer. It may be part of a combination. Combinations of these drugs may be drug combinations used in chemotherapy, including EOX chemotherapy, ECF chemotherapy, ECX chemotherapy, EOF chemotherapy, FLO chemotherapy, FOLFOX chemotherapy, FOLFIRI chemotherapy, DCF chemotherapy and FLOT. It may be a drug combination such as that used in chemotherapy selected from the group consisting of chemotherapy.

EOX The drug combination used in chemotherapy consists of epirubicin, oxaliplatin, and capecitabine. The drug combination used in ECF chemotherapy consists of epirubicin, cisplatin, and 5-fluorouracil. ECX The drug combination used in chemotherapy consists of epirubicin, cisplatin, and capecitabine. EOF The drug combination used in chemotherapy consists of epirubicin, oxaliplatin, and 5-fluorouracil.

Epirubicin is usually given at a dose of 50 mg/m2, cisplatin at a dose of 60 mg/m2, oxaliplatin at a dose of 130 mg/m2, and 5-fluorouracil at a dose of 200 mg/m2/day. , Oral capecitabine is administered by prolonged intravenous infusion (protracted venous infusion) at a dose of 625 mg/m2 twice a day for a total of 8 times in a 3-week cycle.

FLO The drug combination used in chemotherapy is 5-fluorouracil, folinic acid and oxaliplatin (usually 5-fluorouracil 2,600 mg/m ² 24-h infusion, folinic acid 200 mg/m ² and oxaliplatin 85 mg/m ² ). (every two weeks).

FOLFOX is a chemotherapy regimen consisting of folinic acid (leucovorin), 5-fluorouracil, and oxaliplatin. The recommended dosing schedule, given every two weeks, is as follows: Day 1: Oxaliplatin 85 mg/m ² IV infusion and leucovorin 200 mg/m ² IV infusion, followed by 5-FU 400 mg/m ² IV bolus, followed by 22 5-FU 600 mg/m ² IV infusion continuously; Day 2: Leucovorin 200 mg/m ² IV infusion over 120 minutes, followed by 5-FU 400 mg/m ² IV bolus over 2-4 minutes, followed by 5-FU 600 mg/m over 22 consecutive hours. ² IV infusion.

The drug combination used in FOLFIRI chemotherapy consists of 5-fluorouracil, leucovorin, and irinotecan.

DCF chemotherapy consists of docetaxel, cisplatin, and 5-fluorouracil.

The drug combination used in FLOT chemotherapy consists of docetaxel, oxaliplatin, 5-fluorouracil, and folinic acid.

“Folinic acid” or “leucovorin” refers to a compound useful in synergistic combination with the chemotherapy agent 5-fluorouracil. Folinic acid has the following structural formula:

In particular, this term refers to the compound (2S)-2-{[4-[(2-amino-5-formyl-4-oxo-5,6,7,8-tetrahydro-1H-pteridine-6- refers to 1) methylamino]benzoyl]amino}pentadiophosphoric acid.

γδ T cells (gamma delta T cells) are a small subset of T cells that bear a unique T cell receptor (TCR) on their surface. The majority of T cells have a TCR consisting of two glycoprotein chains called α- and β-TCR chains. In contrast, in γδ T cells, the TCR consists of one γ-chain and one δ-chain. This group of T cells is usually very rare compared to αβT cells. Human γδ T cells play an important role in stress-surveillance responses such as infectious diseases and autoimmunity. It is inferred that transformation-induced changes in tumors also trigger a stress-surveillance response mediated by γδ T cells, thereby enhancing antitumor immunity. After antigen engagement, activated γδ T cells in the lesion provide chemokines and/or cytokines (e.g. INFγ, TNFα) that mediate the recruitment of other effector cells and cytotoxicity (via dead receptors and cytolytic granulocyte pathways). It is very important to represent immediate effector functions such as ADCC.

Most γδ T cells in peripheral blood express the Vγ9Vδ2 T cell receptor (TCRγδ). Vγ9Vδ2 T cells are found only in humans and primates and are believed to play an early and important role in sensing “danger” from invading pathogens as they expand rapidly in many acute infections, such as tuberculosis, salmonellosis, ehrlichiosis, brucellosis, and tularemia. , listeriosis, toxoplasmosis and malaria, can surpass all other lymphocytes within a few days.

γδ T cells respond to small non-peptidyl phosphorylated antigens (phosphoantigens) such as isopentenyl pyrophosphate (IPP) produced in mammalian cells via the mevalonate pathway and pyrophosphate synthesized in bacteria. react. While IPP production in normal cells is insufficient for activation of γδ T cells, dysregulation of the mevalonate pathway in tumor cells leads to accumulation of IPP and activation of γδ T cells. IPPs can also be increased therapeutically by aminobisphosphonates, which inhibit the mevalonate pathway enzyme farnesyl pyrophosphate synthase (FPPS). Among them, zoledronic acid (ZA, zoledronate, Zometa ^™ , Novartis) represents such aminobisphosphonates, and is already being administered clinically to patients for the treatment of osteoporosis and metastatic bone disease. When treating PBMCs in vitro, ZA is specifically taken up by monocytes. IPP accumulates in monocytes and differentiates into antigen-presenting cells that stimulate the development of γδ T cells. In this setting, the addition of interleukin-2 (IL-2) as a growth and survival factor for activated γδ T cells is desirable. Finally, certain alkylated amines have been described to activate Vγ9Vδ2 T cells in vitro, but only at the level of millimolar concentrations.

According to the present invention, the term "agent that stimulates γδ T cells" refers to an agent that stimulates the development of γδ T cells, in particular Vγ9Vδ2 T cells, in vitro and/or in vivo, particularly by inducing the activation and expansion of γδ T cells. It relates to compounds that This term refers to isopentenyl pyrophosphate (IPP) produced in mammalian cells in vitro and/or in vivo, preferably by inhibiting the mevalonate pathway enzyme farnesyl pyrophosphate synthase (FPPS). It relates to compounds that increase

A particular group of compounds that stimulate γδ T cells are bisphosphonates, especially nitrogen-containing bisphosphonates (N-bisphosphonates; aminobisphosphonates).

For example, bisphosphonates suitable for use in the present invention may include one or more of the following compounds, analogs and derivatives, pharmaceutical salts, hydrates, esters, conjugates and prodrugs:

[1-hydroxy-2-(1H-imidazol-1-yl)ethane-1,1-diyl]bis(phosphonic acid), zoledronic acid, such as zoledronate;

(dichloro-phosphono-methyl)phosphonic acid, such as clodronate

{1-hydroxy-3-[methyl(pentyl)amino]propane-1,1-diyl}bis(phosphonic acid), ibandronic acid, such as ibandronate

(3-amino-1-hydroxypropane-1,1-diyl)bis (phosphonic acid), pamidronic acid, such as pamidronate

(1-hydroxy-1-phosphono-2-pyridin-3-yl-ethyl)phosphonic acid, risedronic acid such as risedronate;

(1-hydroxy-2-imidazo[1,2-a]pyridin-3-yl-1-phosphonoethyl)phosphonic acid, minodronic acid;

*[3-(dimethylamino)-1-hydroxypropane-1,1-diyl]bis(phosphonic acid), olpadronic acid.

[4-Amino-1-hydroxy-1-(hydroxy-oxido-phosphoryl)-butyl]phosphonic acid, aledronic acid, such as aledronate;

[(cycloheptylamino)methylene]bis(phosphonic acid), incadronic acid;

(1-hydroxyethane-1,1-diyl)bis(phosphonic acid), etidronic acid such as etidronate; and

{[(4-chlorophenyl)thio]methylene}bis(phosphonic acid), tiludronic acid.

According to the present invention, zoledronic acid (INN) or zoledronate (marketed by Novartis under the trade names Zometa, Zomera, Aclasta and Reclast) are particularly preferred bisphosphonates. Zometa is used to prevent skeletal fractures and treat osteoporosis in patients with cancers such as multiple myeloma and prostate cancer. It can also be used to treat hypercalcemia of malignancy and may be helpful in treating pain caused by bone metastases.

In one preferred embodiment, the agent that stimulates γδ T cells according to the invention is administered in combination with IL-2. This combination was shown to be particularly effective in mediating expansion and activation of γ9δ2 T cells.

Interleukin-2 (IL-2) is an interleukin, a type of cytokine signaling molecule in the immune system. This is a protein that attracts lymphocytes and forms part of our body's natural response to microbial infection, distinguishing between our body (self) and foreign agents (non-self). IL-2 mediates its effects by binding to the IL-2 receptor expressed by lymphocytes.

The IL-2 used in the present invention may be any IL-2 that supports or enables stimulation of γδ T cells, and may be derived from any species, preferably from humans. IL-2 may be isolated, recombinantly produced, synthetic IL-2, naturally occurring or modified IL-2.

In one embodiment of the invention, standard chemotherapy according to the EOX regimen is administered over up to 8 cycles in combination with an antibody with the ability to bind CLDN18.2, especially IMAB362. Dosage and administration schedule can be carried out as follows:

● During the EOX phase, 50 mg/m2 epirubicin i.v. as a 15-minute infusion on day 1 of each cycle. Administer.

● During the EOX phase, 130 mg/m2 oxaliplatin was administered i.v. as a 2h infusion on day 1 of each cycle. Administer.

● During the EOX phase, starting in the evening of Day 1 of each cycle, 625 mg/m2 capecitabine p.o. twice daily, morning and evening, for 21 days. Administer.

● On Day 1 of Cycle 1, 1000 mg/m2 antibody was administered i.v. as a 2h infusion. Administer. Thereafter, after completion of oxaliplatin infusion, 600 mg/m2 antibody was injected i.v. for 2h on the first day of each cycle. Administer.

● After completion of chemotherapy, patients continue to receive 600 mg/m2 antibody as a 2h infusion every 3 or 4 weeks.

In one embodiment of the invention, standard chemotherapy according to the EOX regimen is administered for up to 8 cycles (24 weeks) in combination with an antibody with the ability to bind ZA/IL-2 and CLDN18.2, especially IMAB362.

The term “antigen” refers to an agent, such as a protein or peptide, that contains an epitope against which an immune response is or will be directed. In a preferred embodiment, the antigen is a tumor-related antigen, such as CLDN18.2, i.e. a member of cancer cells that can be derived from the cytoplasm, cell surface and cell nucleus, especially as a surface antigen on cancer cells or produced in large quantities intracellularly. Those that are antigens are preferred.

In the context of the present invention, a “tumor-related antigen” is preferably expressed specifically or at a specific stage of development in a limited number of tissues and/or organs under normal conditions and is expressed in one or more tumors or cancerous tissues or is abnormal. It relates to proteins expressed as In the context of the present invention, tumor-related antigens are preferably associated with the cell surface of cancer cells and are not expressed in normal tissues or are expressed only rarely.

The term “epitope” refers to an antigenic determinant within a molecule, i.e., a portion of a molecule that is recognized by the immune system, such as by an antibody. For example, an epitope is a discrete, three-dimensional portion of an antigen that is recognized by the immune system. Epitopes usually constitute chemically active surface groupings of molecules, such as amino acids or sugar side chains, and usually have specific three-dimensional structural features and specific charge characteristics.

Conformational epitopes and non-conformational epitopes are distinguished in that binding to the former is lost in the presence of a denaturing solvent, whereas binding to the latter is not. The epitope of a protein such as CLDN18.2 preferably comprises a continuous or discontinuous portion of the protein and is preferably e.g. 5 to 100, preferably 5 to 50, more preferably 8 to 30, most preferably 8 to 30. It is preferably 10 to 25 amino acids, for example, the epitope length is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24. , or preferably 25 amino acids.

“Antibody” refers to a glycoprotein containing at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, and includes any molecule containing an antigen-binding portion thereof. The term “antibody” includes monoclonal antibodies and antibody fragments or antibody derivatives, including human antibodies, humanized antibodies, chimeric antibodies, single chain antibodies such as scFv's and antigen-binding antibody fragments such as Fab and Fab' fragments. This includes, but is not limited to, all recombinant forms of antibodies, such as antibodies expressed in prokaryotes, aglycosylated antibodies, and all antigen-binding antibody fragments and derivatives described herein. Each heavy chain consists of a heavy chain variable region (abbreviated as VH) and a heavy chain constant region. Each light chain consists of a light chain variable region (abbreviated as VL) and a light chain constant region. The VH and VL regions can be further subdivided into hypervariable regions, termed complementarity determining regions (CDRs), and interspersed with more conservative regions, termed framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

Antibodies described herein may be human antibodies. As used herein, the term “human antibody” is intended to encompass antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies described herein may contain amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations induced in vitro by random or site-directed mutagenesis or somatic mutations in vivo). ).

“Humanized antibody” refers to a molecule in which the antigen-binding portion of the molecule is substantially derived from an immunoglobulin from a non-human species, while the remainder of the immunoglobulin structure of the molecule is based on the structure and/or sequence of a human immunoglobulin. The antigen-binding portion may comprise the complete variable domain fused onto a constant domain or may comprise only complementarity determining regions (CDRs) grafted to appropriate framework portions within the variable domain. The antigen-binding portion may be wild type or may be modified by one or more amino acid substitutions, such as to more closely resemble a human immunoglobulin. Some types of humanized antibodies preserve all CDR sequences (such as humanized mouse antibodies containing all six CDRx from a mouse antibody). Other types may differ from the original antibody in one or more CDRs.

The term "chimeric antibody" means that portions of the respective amino acid sequences of the heavy and light chains are homologous to corresponding sequences in antibodies belonging to or derived from a particular species, while the remaining segments of the chain is otherwise homologous to the corresponding sequences. Typically, the variable regions of the light and heavy chains resemble the variable regions of antibodies derived from one species of mammal, while the constant regions are homologous to sequences of antibodies derived from another species. One distinct advantage of such chimeric forms is that the variable regions can be conveniently derived from currently known sources, for example, using readily available B-cells or hybridomas from non-human host organisms in combination with constant regions derived from human cell production. That there is. While the variable region has the advantage of being individually prepared and specificity is not affected by the source, the constant region, which is human, elicits no better immune response from human subjects when antibodies are injected than the constant region from a non-human source. . However, the definition is not limited to these particular examples.

The term “antigen-binding portion” of an antibody (or simply, “binding portion”) or “antigen-binding fragment” (or simply, “binding fragment”) of an antibody refers to a member of an antibody that retains the ability to specifically bind to the antibody. Refers to the above fragments. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments included within the term "antigen-binding portion" of an antibody are ( i) Fab fragments, monovalent fragments consisting of VL, VH, CL and CH domains; (ii) F(ab') ₂ fragments, 2 comprising two Fab fragments connected by a disulfide bond in the hinge region (iii) Fd fragments consisting of the VH and CH domains; (iv) Fv fragments consisting of the VL and VH domains of a single arm of the antibody, (v) dAb fragments (Ward et al. (1989) Nature 341: 544-546), consisting of a VH domain; (vi) separate complementarity determining regions (CDRs), and (vii) combinations of two or more CDRs that can be optionally joined by a synthetic linker. Additionally, although the two domains, VL and VH, are encoded by separate genes, the VL and VH regions are linked to a synthetic linker that allows them to form a single protein chain that pairs to form monovalent molecules. (known as single chain Fv (scFv); Bird et al (1988) Science 242: 423-426; and Huston et al (1988) Proc. Natl. Acad. Sci. USA 85: 5879 (see -5883). It is believed that such single chain antibodies may be included within the term "antigen-binding fragment" of an antibody. Additional examples include (i) a binding domain polypeptide fused to an immunoglobulin hinge region polypeptide, ( These are binding-domain immunoglobulin fusion proteins that include (ii) an immunoglobulin heavy chain CH2 constant region fused to the hinge region, and (iii) an immunoglobulin heavy chain CH3 constant region fused to the CH2 constant region. The binding domain polypeptide may be a heavy chain variable region or a light chain variable region. Binding-domain immunoglobulin fusion proteins are additionally disclosed in US 2003/0118592 and US 2003/0133939. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for use in the same manner as intact antibodies.

A “bispecific molecule” is intended to include any agent, such as a protein, peptide or protein or peptide complex, which has two different binding specificities. For example, a molecule can bind or interact with (a) a cell surface antigen, and (b) an Fc receptor on the surface of an effector cell. The term “multispecific molecule” or “heterospecific molecule” is intended to include any agent, such as a protein, peptide or protein or peptide complex, which has two or more different binding specificities. For example, the molecule can bind or interact with (a) a cell surface antigen, (b) an Fc receptor on the surface of an effector cell, and (c) at least one other component. Accordingly, the present invention includes, but is not limited to, bispecific, trispecific, quadruple specific, and other multispecific molecules directed to CLD18.2 and other targets including Fc receptors on effector cells. The term “bispecific antibodies” also includes diabodies. The variants are bivalent, and the VH and VL domains are expressed in a single polypeptide chain, but use a linker that is too short to allow pairing between the two domains on the same chain, thereby allowing the domains to form complementary domains on other chains. These are bispecific antibodies that can pair with and create two antigen binding sites (Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448; Poljak, R. J., et al. ( 1994) Structure 2: 1121-1123).

Antibodies may also be conjugated to therapeutic moieties or agents, such as cytotoxins, drugs (eg, immunosuppressants), or radioisotopes. A cytotoxin or cytotoxic agent includes any agent that is harmful to cells or specifically kills cells. Examples include taxol, cytochalasin B, gramicidin D, ithidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, and daunoru. Vicin, dihydroxyanthracene dione, mitoxantrone, mithramycin, actinomycin D, 1-dihydroxytestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and their analogs or congeners. Contains agonists. Suitable therapeutic agents for forming antibody conjugates include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5 -fluorouracil decarbazine), alkylating agents (e.g., macrorethamine, thioepa, chlorambucil, melphalan, carmustine (BSNU), and lomustine (CCNU), cyclophosphamide, busulfan , dibromomannitol, streptozotocin, mitomycin C and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (such as daunorubicin (formerly daunomycin)) and doxorubicin), Antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC), and anti-mitotic agents (e.g., vincristine and vinblastine). Preferred In one embodiment, the therapeutic agent is a cytotoxic agent or a radiotoxic agent.In another embodiment, the therapeutic agent is an immunosuppressant.In another embodiment, the therapeutic agent is GM-CSF.In a preferred embodiment, the therapeutic agent is doxorubicin, cisplatin. , bleomycin, sulfate, carmustine, chlorambucil, cyclophosphamide or ricin A.

Antibodies can also bind to radioactive isotopes such as iodine-131, yttrium-90, and indium-111, producing cytotoxic radiopharmaceuticals.

The antibody conjugates of the invention can be used to modify the biological response of interest, and the drug moiety is not intended to be limited to traditional chemotherapy agents. For example, the drug moiety may be a protein or polypeptide that has a desired biological action. Such proteins include, for example, the enzymatically active toxin, or its active fragments, abrin, ricin A, Pseudomonas exotoxin, diphtheria toxin; Proteins such as necrosis factor or interferon-γ; or, for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulosa macrophage colony-stimulating factor (GM- CSF), granular colony stimulating factor (G-CSF) or other growth factors.

Techniques for conjugating these therapeutic moieties to antibodies are well known. See, for example, Arnon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy”, in Monoclonal Antibodies and Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985) ; Hellstrom et al., "Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", in Monoclonal Antibodies '84: Biological and Clinical Applications, Pinchera et al. (eds. ), pp. 475-506 (1985); "Analysis, Results, and Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal Antibodies For Cancer Detection and Therapy, Baldwin et al (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., "The Preparation and Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol Rev., 62: 119-58 (1982).

In the present invention, when an antibody is obtained from a system by immunizing an animal or screening an immunoglobulin gene library, the antibody is “derived from” a specific germline sequence, and the selected antibody is derived from that germline immunoglobulin gene. is at least 90% identical, more preferably at least 95% identical, and even more preferably at least 96%, 97%, 98%, or 99% identical to the coding amino acid sequence. In general, an antibody derived from a specific germline sequence has no more than 10 amino acid differences from the amino acid sequence encoded by the germline immunoglobulin, more preferably no more than 5 amino acids, and even more preferably 4, 3, or 2 amino acid differences. It is preferable that there are 1 or less.

In the present invention, the term “heteroantibody” means that at least two of two or more antibodies, derivatives thereof, or antigen-binding portions bound together have different specificities. These different specificities include binding specificities for Fc receptors on effector cells, and binding specificities for antigens or epitopes on target cells, such as tumor cells.

Antibodies described herein may be monoclonal antibodies. As used herein, the term “monoclonal antibody” refers to a preparation of antibody molecules of single molecular composition. Monoclonal antibodies exhibit single binding specificity and affinity. In one embodiment, monoclonal antibodies are produced by hybridomas comprising B cells obtained from non-human animals, such as mice, fused to immortalized cells.

Antibodies described herein may be recombinant antibodies. In the present invention, the term "recombinant antibody" refers to (a) antibodies isolated from animals (e.g., mice) that are transgenic or transgenic with respect to immunoglobulin genes or hybridomas produced therefrom, (b) antibodies. (c) antibodies isolated from host cells transformed to express, e.g., transfectoma, (c) antibodies isolated from recombinant, combinatorial antibody libraries, and (d) splices of immunoglobulin gene sequences to other DNA sequences. It includes all antibodies isolated, generated, expressed or prepared by recombinant means, including antibodies isolated, generated, expressed or prepared by other means related to the antibody.

Antibodies described herein may be derived from different species, including, but not limited to, mouse, rat, rabbit, guinea pig, and human.

Antibodies described herein include polyclonal and monoclonal antibodies and include IgA antibodies such as IgA1 or IgA2, IgG1, IgG2, IgG3, IgG4, IgE, IgM, and IgD. In various embodiments, the antibody is an IgG1 antibody, more particularly an IgG1, kappa or IgG1, lambda isotype (i.e. IgG1, κ, λ), an IgG2a antibody (e.g. IgG2a, κ, λ), an IgG2b antibody (e.g. IgG2b, κ, λ), IgG3 antibodies (eg IgG3, κ, λ) or IgG4 antibodies (eg IgG4, κ, λ).

As used herein, the term “transfectoma” refers to a cell that expresses an antibody, including CHO cells, NS/0 cells, HEK293 cells, HEK293T cells, plant cells, or fungi, including yeast. Includes recombinant eukaryotic host cells.

In the present invention, “heterologous antibody” is defined in relation to a transformed organism that produces such antibody. This term refers to an antibody that has an amino acid sequence or encodes a nucleic acid sequence corresponding to that occurring in an organism that does not constitute a transformed organism, and that is generally derived from species other than the transformed organism.

As used herein, “heterohybrid antibody” refers to an antibody that has light and heavy chains from different organisms. For example, an antibody that has a human heavy chain associated with a murine light chain is a heterohybrid antibody.

The present invention includes all antibodies and derivatives of antibodies as described herein, the object of which is encompassed by the term “antibody”. The term “antibody derivative” refers to any modified form of an antibody, such as a conjugate of the antibody with another agent or antibody, or antibody fragment.

Antibodies described in the present invention are preferably isolated. As used herein, the term “isolated antibody” is intended to refer to an antibody that is substantially free of other antibodies with different antigenic specificities (e.g., an isolated antibody that specifically binds to CLDN18.2 may be substantially free of other antibodies of different antigenic specificities). There are no antibodies that specifically bind to antigens other than CLDN18.2). Isolated antibodies that specifically bind to an epitope, isoform or variant of human CLDN18.2, however, have cross-reactivity with other related antigens, e.g., from other species (e.g., CLDN18.2 species homolog). Additionally, the isolated antibody may be substantially free of other cellular and/or chemical agents. In one embodiment of the invention, the combination of “isolated” monoclonal antibodies relates to antibodies having different specificities and combined as a well-defined composition or mixture.

In the present invention, the term “binding” preferably relates to specific binding.

In the present invention, if an antibody has significant affinity for a given target and binds to the given target according to a standard analysis method, the antibody has the ability to bind to the given target. “Affinity” or “binding affinity” is often measured by the equilibrium dissociation constant (K _D ). Preferably, the term "significant affinity" means 10 ^-5 M or less, 10 ^-6 M or less, 10 ^-7 M or less, 10 ^-8 M or less, 10 ^-9 M or less. It means binding to a given target with a dissociation constant (K _D ) value of less than, 10 ^-10 M or less, 10 ^-11 M or less, or 10 ^-12 M or less.

If an antibody does not have significant affinity for the target and does not significantly bind to the target in a standard assay, especially if it does not bind detectably, then the antibody is (substantially) not capable of binding to the target. There is nothing. Preferably, the antibody detectably binds to the target when present at a concentration of at most 2, preferably at most 10, more preferably at most 20, especially at most 50 or 100 μg/ml or more. no. Preferably, if the K _D value is at least 10 times, 100 times, 10 ³ times, 10 ⁴ times, 10 ⁵ times, or 10 ⁶ times greater than the binding K _D for a given target to which the antibody is capable of binding. If it binds highly to the target, the antibody does not have significant affinity for the target. For example, if the binding K _D value of an antibody to a target to which the antibody can bind is 10 ^-7 M, the binding K _D value to a target to which the antibody does not have significant affinity is at least 10 ^-6 M, 10 ^-5 M , 10 ^-4 M, 10 ^-3 M, 10 ^-2 M, or 10 ^-1 M.

If an antibody is unable to bind to another target, i.e., has no significant affinity for another target, and does not significantly bind to another target in standard assays, but is capable of binding to a given target. , the antibody is specific to the given target. In the present invention, if an antibody can bind to CLDN18.2 but cannot (substantially) bind to other targets, the antibody is specific for CLDN18.2. Preferably, the affinity and binding to another target is to a protein unrelated to CLDN18.2, such as bovine serum albumin (BSA), casein, human serum albumin (HSA), or an MHC molecule or transferrin. The antibodies are specific for CLDN18.2 unless they significantly exceed the affinity or binding to non-claudin transmembrane proteins, such as receptors, or to other specific polypeptides. Preferably, the K _D value for binding to a target for which an antibody is not specific is at least 10-fold, 100-fold, 10 ³ -fold, 10 ⁴ -fold, 10 ⁵ -fold, or 10 ⁶ -fold higher. If it binds to a given target with a low K _D value, the antibody is specific for that target. For example, if an antibody has a K _D value of 10 ^-7 M for binding to a specific target, the K _D value for binding to a non-specific target is at least 10 ^-6 M, 10 ^-5 M, It may be 10 ^-4 M, 10 ^-3 M, 10 ^-2 M, or 10 ^-1 M.

Binding of an antibody to its target can be determined experimentally using any suitable method, see, for example, Berzofsky et al., "Antibody-Antigen Interactions" In Fundamental Immunology, Paul, WE, Ed., Raven Press New York, NY (1984), Kuby, Janis Immunology, WH Freeman and Company New York, NY (1992)) and the methods described therein. Use of balanced dialysis; Use of the BIAcore 2000 instrument using the general procedures outlined by the manufacturer; by radioimmunoassay using radiolabeled target antigens; Alternatively, affinity can be readily determined using routine techniques, such as by other methods known to those skilled in the art. Affinity data can be analyzed, for example, by the method of Scatchard et al., Ann NY Acad. ScL, 51:660 (1949). If measured under different conditions (e.g. salt concentration, pH), the measured affinity of a particular antibody-antigen binding may vary. Therefore, measurements of affinity and other antigen-binding parameters such as K _D and IC ₅₀ are preferably made using standardized antibody and antigen solutions and standardized buffers.

In the present invention, “isotype” refers to an antibody class (eg, IgM or IgG1) encoded by heavy chain constant region genes.

As used herein, “isotype switching” refers to the phenomenon by which the class or isotype of antibodies changes from one Ig class to one of the other Ig classes.

As applied to the purpose, the term "naturally occurring" as used herein refers to the fact that the purpose can be found in nature. For example, a naturally occurring polypeptide or polynucleotide sequence that can be isolated from a source in nature and is present in an organism (including viruses) that has not been intentionally modified by humans in the laboratory.

As used herein, the term "rearranged" refers to the arrangement of the heavy or light chain immunoglobulin locus, wherein the V segment is D-J in a structure that encodes essentially a complete VH or VL domain. or positioned immediately adjacent to the J segment. Rearranged immunoglobulin (antibody) gene loci can be identified by comparison with germline DNA; The rearranged locus will have at least one recombined heptamer/nanomer homology element.

As used herein with respect to a V segment, the term "unrearranged" or "germline arrangement" refers to an arrangement in which the V segment is not recombined and is directly adjacent to a D or J segment.

In the present invention, the antibody capable of binding to CLDN18.2 is an epitope present in CLDN18.2, preferably an epitope located within the extracellular domains of CLDN18.2, especially the first extracellular domain, particularly CLDN18.2. It is an antibody that can bind to the epitope present within amino acid positions 29 to 78. In certain embodiments, an antibody capable of binding to CLDN18.2 (i) epitopes on CLDN18.2, preferably SEQ ID NO: 3, 4, and 5, that are not present on CLDN18.1, ( ii) an epitope located on CLDN18.2-Loop1, preferably on SEQ ID NO: 8, (iii) an epitope located on CLDN18.2-Loop2, preferably on SEQ ID NO: 10, (iv) CLDN18.2-LoopD3, preferably an epitope located on SEQ ID NO: 11, (v) an epitope encompassing CLDN18.2-Loop1 and CLDN18.2-LoopD3, or (vi) CLDN18.2- It is an antibody capable of binding to the non-glycosylated epitope located on LoopD3, preferably SEQ ID NO:9.

According to the present invention, an antibody capable of binding to CLDN18.2 is an antibody that does not bind to CLDN18.1 but has the ability to bind to CLDN18.2. Preferably, the antibody having the ability to bind CLDN18.2 is specific for CLDN18.2. Preferably, the antibody capable of binding to CLDN18.2 is preferably an antibody capable of binding to CLDN18.2 expressed on the surface of the cell. In a particularly preferred embodiment, the antibody capable of binding to CLDN18.2 binds to a native epitope of CLDN18.2 present on the surface of living cells. Preferably, the antibody capable of binding CLDN18.2 binds one or more peptides selected from the group consisting of SEQ ID NOs: 1, 3-11, 44, 46, and 48-50. Preferably, the antibody capable of binding to CLDN18.2 is specific for the above-mentioned protein, peptide or immunogenic fragment or derivative thereof. Antibodies capable of binding to CLDN18.2 are proteins or peptides comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3-11, 44, 46, and 48-50, or expressing said proteins or peptides. It can be obtained by a method comprising immunizing an animal with a nucleic acid or host cell. Preferably, the antibody binds to cancer cells, especially cancer cells of the types described above, and does not substantially bind to non-cancerous cells.

Preferably, binding of an antibody capable of binding CLDN18.2 to a cell expressing CLDN18.2 induces or mediates death of the cell expressing CLDN18.2. The cells expressing CLDN18.2 are preferably cancer cells, especially tumorigenic cancer cells of the stomach, esophagus, pancreas, lung, ovary, colon, liver, head and neck, and gallbladder. Preferably, the antibody induces one or more of complement-dependent cytotoxicity (CDC)-mediated lysis, antibody-dependent cytotoxicity (ADCC)-mediated lysis, apoptosis, and inhibition of proliferation of cells expressing CLDN18.2. Induces or mediates death. Preferably, ADCC-mediated lysis of cells occurs, in certain embodiments, in the presence of effector cells selected from the group consisting of monocytes, mononuclear cells, NK cells, and PMNs. Inhibition of cell proliferation can be measured in vitro by detecting cell proliferation in an assay using bromodeoxyuridine (5-bromo-2-deoxyuridine, BrdU). BrdU is a synthetic nucleoside that is an analog of thymidine and can be incorporated into the newly synthesized DNA of replicating cells (during the S phase of the cell cycle), displacing thymidine during DNA replication. For example, antibodies specific for BrdU can be used to detect incorporated chemical agents, thereby detecting cells that are actively replicating their DNA.

In preferred embodiments, the antibodies described herein may be characterized by one or more of the following properties:

a) Specificity for CLDN18.2;

b) a binding specificity for CLDN18.2 of less than or equal to about 100 nM, preferably less than or equal to about 5-10 nM and, more preferably, less than or equal to about 1-3 nM,

c) the ability to induce or mediate CDC on CLDN18.2 positive cells;

d) Ability to induce or mediate ADCC on CLDN18.2 positive cells;

e) Ability to inhibit the growth of CLDN18.2 positive cells;

f) Ability to induce apoptosis of CLDN18.2 positive cells.

In a particularly preferred embodiment, the antibody capable of binding to CLDN18.2 has been deposited with DSMZ (Mascheroder Weg 1b, 31824 Braunschweig, Germany; new address: Inhoffenstr. 7B, 31824 Braunschweig, Germany) and has the following name and accession number: Produced by endowed hybridomas:

a. 182-D1106-055, accession number DSM ACC2737, deposited on October 19, 2005.

b. 182-D1106-056, accession number DSM ACC2738, deposited on October 19, 2005.

c. 182-D1106-057, accession number DSM ACC2739, deposited on October 19, 2005.

d. 182-D1106-058, accession number DSM ACC2740, deposited on October 19, 2005.

e. 182-D1106-059, accession number DSM ACC2741, deposited on October 19, 2005.

f. 182-D1106-062, accession number DSM ACC2742, deposited on October 19, 2005;

g. 182-D1106-067, accession number DSM ACC2743, deposited on October 19, 2005.

h. 182-D758-035, accession number DSM ACC2745, deposited on November 17, 2005.

i. 182-D758-036, accession number DSM ACC2746, deposited on November 17, 2005.

j. 182-D758-040, accession number DSM ACC2747, deposited on November 17, 2005.

k. 182-D1106-061, accession number DSM ACC2748, deposited on November 17, 2005.

*l. 182-D1106-279, accession number DSM ACC2808, deposited on October 26, 2006.

m. 182-D1106-294, accession number DSM ACC2809, deposited on October 26, 2006;

n. 182-D1106-362, accession number DSM ACC2810, deposited on October 26, 2006.

Preferred antibodies according to the invention are those produced by or obtainable from the hybridomas described above; That is, in the case of 182-D1106-055, 37H8, 182-D1106-056 for 182-D1106-056, 38G5, 182-D1106-058 for 182-D1106-057, 38H3, 182-D1106-059 for 39F11, 182-D1106 for 182-D1106-059 43A11 for -062, 61C2 for 182-D1106-067, 26B5 for 182-D758-035, 26D12 for 182-D758-036, 28D10 for 182-D758-040, 42E12 for 182-D1106-061 , 125E1 for 182-D1106-279, 163E12 for 182-D1106-294, and 175D10 for 182-D1106-362; and chimeric and humanized forms thereof.

Preferred chimeric antibodies and their sequences are shown in the following table.

In a preferred embodiment, the antibody, especially the chimeric form of the antibody according to the invention, has a heavy chain constant region (CH) comprising an amino acid sequence derived from a human heavy chain constant region, such as the amino acid sequence shown in SEQ ID NO: 13 or fragments thereof. It is good to include antibodies containing. In another preferred embodiment, the antibody, especially the chimeric form of the antibody according to the invention, comprises an amino acid sequence derived from a human light chain constant region, such as the amino acid sequence shown in SEQ ID NO: 12 or a fragment thereof ( It is recommended to include antibodies containing CL). In a particularly preferred embodiment, the antibody, in particular the chimeric form of the antibody according to the invention, comprises CH or SEQ ID NO: 12 comprising an amino acid sequence derived from human CH, such as the amino acid sequence shown in SEQ ID NO: 13 or a fragment thereof. CL comprising an amino acid sequence derived from human CL, such as the amino acid sequence represented by or fragment thereof.

In one embodiment, the antibody capable of binding to CLDN18.2 includes kappa, murine light chain variable region, human kappa light chain constant region allotype Km(3), murine heavy chain variable region, human IgG1 constant region, It is a chimeric mouse/human IgG1 monoclonal antibody containing the allogeneic G1m(3).

In certain preferred embodiments, the chimeric form of the antibodies comprises a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 15, 16, 17, 18, 19, and fragments thereof : Antibodies comprising a light chain comprising an amino acid sequence selected from the group consisting of 20, 21, 22, 23, 24, 25, 26, 27, 28, and fragments thereof are included.

In certain preferred embodiments, chimeric forms of antibodies include antibodies comprising a combination of heavy and light chains selected from the following possibilities (i) to (ix):

(i) the heavy chain comprises the amino acid sequence represented by SEQ ID NO: 14 or a fragment thereof and the light chain comprises the amino acid sequence represented by SEQ ID NO: 21 or a fragment thereof,

(ii) the heavy chain comprises the amino acid sequence represented by SEQ ID NO: 15 or a fragment thereof and the light chain comprises the amino acid sequence represented by SEQ ID NO: 20 or a fragment thereof,

(iii) the heavy chain comprises the amino acid sequence represented by SEQ ID NO: 16 or a fragment thereof and the light chain comprises the amino acid sequence represented by SEQ ID NO: 22 or a fragment thereof,

(iv) the heavy chain comprises the amino acid sequence represented by SEQ ID NO: 18 or a fragment thereof and the light chain comprises the amino acid sequence represented by SEQ ID NO: 25 or a fragment thereof,

(v) the heavy chain comprises the amino acid sequence represented by SEQ ID NO: 17 or a fragment thereof and the light chain comprises the amino acid sequence represented by SEQ ID NO: 24 or a fragment thereof,

(vi) the heavy chain comprises the amino acid sequence represented by SEQ ID NO: 19 or a fragment thereof and the light chain comprises the amino acid sequence represented by SEQ ID NO: 23 or a fragment thereof,

(vii) the heavy chain comprises the amino acid sequence represented by SEQ ID NO: 19 or a fragment thereof and the light chain comprises the amino acid sequence represented by SEQ ID NO: 26 or a fragment thereof,

(viii) the heavy chain comprises the amino acid sequence represented by SEQ ID NO: 19 or a fragment thereof and the light chain comprises the amino acid sequence represented by SEQ ID NO: 27 or a fragment thereof, and

(ix) the heavy chain comprises the amino acid sequence represented by SEQ ID NO: 19 or a fragment thereof and the light chain comprises the amino acid sequence represented by SEQ ID NO: 28 or a fragment thereof.

As described above, in the present invention, “fragment” or “fragment of amino acid sequence” refers to a part of an antibody sequence, that is, an antibody sequence shortened at the N- and/or C-terminus, which refers to the above-mentioned antibody sequence. When replacing an antibody sequence, it refers to a sequence that maintains the binding of the antibody to CLDN18.2 and preferably maintains the function of the antibody as described above, such as CDC-mediated lysis or ADCC-mediated lysis ability. . Preferably, the fragment of amino acids comprises at least 80%, preferably at least 90%, 95%, 96%, 97%, 98%, or 99% of the amino acid residues of the amino acid sequence. SEQ ID NOs: The fragment of amino acid sequence selected from the group consisting of 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, and 28 is preferably N- It relates to the above sequence with 17, 18, 19, 20, 21, 22 or 23 amino acids removed from the terminus.

In a preferred embodiment, the antibody capable of binding to CLDN18.2 has a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 29, 30, 31, 32, 33, 34, and fragments thereof ( Includes VH).

In a preferred embodiment, the antibody capable of binding to CLDN18.2 has an amino acid sequence selected from the group consisting of SEQ ID NO: 35, 36, 37, 38, 39, 40, 41, 42, 43, and fragments thereof. Comprising a light chain variable region (VL).

In certain preferred embodiments, the antibody capable of binding CLDN18.2 comprises a combination of a heavy chain variable region (VH) and a light chain variable region (VL) selected from the following possible instances (i) to (ix):

(i) VH comprises the amino acid sequence represented by SEQ ID NO: 29 or a fragment thereof and VL comprises the amino acid sequence represented by SEQ ID NO: 36 or a fragment thereof,

(ii) VH comprises the amino acid sequence represented by SEQ ID NO: 30 or a fragment thereof and VL comprises the amino acid sequence represented by SEQ ID NO: 35 or a fragment thereof,

(iii) VH comprises the amino acid sequence represented by SEQ ID NO: 31 or a fragment thereof and VL comprises the amino acid sequence represented by SEQ ID NO: 37 or a fragment thereof,

(iv) VH comprises the amino acid sequence represented by SEQ ID NO: 33 or a fragment thereof and VL comprises the amino acid sequence represented by SEQ ID NO: 40 or a fragment thereof,

(v) VH comprises the amino acid sequence represented by SEQ ID NO: 32 or a fragment thereof and VL comprises the amino acid sequence represented by SEQ ID NO: 39 or a fragment thereof,

(vi) VH comprises the amino acid sequence represented by SEQ ID NO: 34 or a fragment thereof and VL comprises the amino acid sequence represented by SEQ ID NO: 38 or a fragment thereof,

(vii) VH comprises the amino acid sequence represented by SEQ ID NO: 34 or a fragment thereof and VL comprises the amino acid sequence represented by SEQ ID NO: 41 or a fragment thereof,

(viii) VH comprises the amino acid sequence represented by SEQ ID NO: 34 or a fragment thereof and VL comprises the amino acid sequence represented by SEQ ID NO: 42 or a fragment thereof,

(ix) VH comprises the amino acid sequence represented by SEQ ID NO: 34 or a fragment thereof and VL comprises the amino acid sequence represented by SEQ ID NO: 43 or a fragment thereof.

In one preferred embodiment, the antibody capable of binding CLDN18.2 comprises a VH comprising a set of complementarity-determining regions CDR1, CDR2 and CDR3 selected from the following embodiments (i) to (vi):

(i) CDR1: positions 45-52 of SEQ ID NO: 14, CDR2: positions 70-77 of SEQ ID NO: 14, CDR3: positions 116-125 of SEQ ID NO: 14,

(ii) CDR1: positions 45-52 of SEQ ID NO: 15, CDR2: positions 70-77 of SEQ ID NO: 15, CDR3: positions 116-126 of SEQ ID NO: 15,

(iii) CDR1: positions 45-52 of SEQ ID NO: 16, CDR2: positions 70-77 of SEQ ID NO: 16, CDR3: positions 116-124 of SEQ ID NO: 16,

(iv) CDR1: positions 45-52 of SEQ ID NO: 17, CDR2: positions 70-77 of SEQ ID NO: 17, CDR3: positions 116-126 of SEQ ID NO: 17,

(v) CDR1: positions 44-51 of SEQ ID NO: 18, CDR2: positions 69-76 of SEQ ID NO: 18, CDR3: positions 115-125 of SEQ ID NO: 18, and

(vi) CDR1: positions 45-53 of SEQ ID NO: 19, CDR2: positions 71-78 of SEQ ID NO: 19, CDR3: positions 117-128 of SEQ ID NO: 19.

In one preferred embodiment, the antibody capable of binding CLDN18.2 comprises a VL comprising a set of complementarity-determining regions CDR1, CDR2 and CDR3 selected from the following embodiments (i) to (ix):

(i) CDR1: positions 47-58 of SEQ ID NO: 20, CDR2: positions 76-78 of SEQ ID NO: 20, CDR3: positions 115-123 of SEQ ID NO: 20,

(ii) CDR1: positions 49-53 of SEQ ID NO: 21, CDR2: positions 71-73 of SEQ ID NO: 21, CDR3: positions 110-118 of SEQ ID NO: 21,

(iii) CDR1: positions 47-52 of SEQ ID NO: 22, CDR2: positions 70-72 of SEQ ID NO: 22, CDR3: positions 109-117 of SEQ ID NO: 22,

(iv) CDR1: positions 47-58 of SEQ ID NO: 23, CDR2: positions 76-78 of SEQ ID NO: 23, CDR3: positions 115-123 of SEQ ID NO: 23,

(v) CDR1: positions 47-58 of SEQ ID NO: 24, CDR2: positions 76-78 of SEQ ID NO: 24, CDR3: positions 115-123 of SEQ ID NO: 24,

(vi) CDR1: positions 47-58 of SEQ ID NO: 25, CDR2: positions 76-78 of SEQ ID NO: 25, CDR3: positions 115-122 of SEQ ID NO: 25,

(vii) CDR1: positions 47-58 of SEQ ID NO: 26, CDR2: positions 76-78 of SEQ ID NO: 26, CDR3: positions 115-123 of SEQ ID NO: 26,

(viii) CDR1: positions 47-58 of SEQ ID NO: 27, CDR2: positions 76-78 of SEQ ID NO: 27, CDR3: positions 115-123 of SEQ ID NO: 27, and

(ix) CDR1: positions 47-52 of SEQ ID NO: 28, CDR2: positions 70-72 of SEQ ID NO: 28, CDR3: positions 109-117 of SEQ ID NO: 28.

In a preferred embodiment, the antibody capable of binding to CLDN18.2 is a combination of VH and VL, each comprising a set of complementarity-determining regions CDR1, CDR2 and CDR3 selected from the following embodiments (i) to (ix) Includes:

(i) VH: CDR1: positions 45-52 of SEQ ID NO: 14, CDR2: positions 70-77 of SEQ ID NO: 14, CDR3: positions 116-125 of SEQ ID NO: 14, VL: CDR1: SEQ ID NO: positions 49-53 of 21, CDR2: SEQ ID NO: positions 71-73 of 21, CDR3: positions 110-118 of SEQ ID NO: 21,

(ii) VH: CDR1: positions 45-52 of SEQ ID NO: 15, CDR2: positions 70-77 of SEQ ID NO: 15, CDR3: positions 116-126 of SEQ ID NO: 15, VL: CDR1: SEQ ID NO: positions 47-58 of 20, CDR2: positions 76-78 of SEQ ID NO: 20, CDR3: positions 115-123 of SEQ ID NO: 20,

(iii) VH: CDR1: positions 45-52 of SEQ ID NO: 16, CDR2: positions 70-77 of SEQ ID NO: 16, CDR3: positions 116-124 of SEQ ID NO: 16, VL: CDR1: SEQ ID NO: positions 47-52 of 22, CDR2: positions 70-72 of SEQ ID NO: 22, CDR3: positions 109-117 of SEQ ID NO: 22,

(iv) VH: CDR1: positions 44-51 of SEQ ID NO: 18, CDR2: positions 69-76 of SEQ ID NO: 18, CDR3: positions 115-125 of SEQ ID NO: 18, VL: CDR1: SEQ ID NO: positions 47-58 of 25, CDR2: positions 76-78 of SEQ ID NO: 25, CDR3: positions 115-122 of SEQ ID NO: 25,

(v) VH: CDR1: positions 45-52 of SEQ ID NO: 17, CDR2: positions 70-77 of SEQ ID NO: 17, CDR3: positions 116-126 of SEQ ID NO: 17, VL: CDR1: SEQ ID NO: positions 47-58 of 24, CDR2: SEQ ID NO: positions 76-78 of 24, CDR3: positions 115-123 of SEQ ID NO: 24,

(vi) VH: CDR1: positions 45-53 of SEQ ID NO: 19, CDR2: positions 71-78 of SEQ ID NO: 19, CDR3: positions 117-128 of SEQ ID NO: 19, VL: CDR1: SEQ ID NO: positions 47-58 of 23, CDR2: SEQ ID NO: positions 76-78 of 23, CDR3: positions 115-123 of SEQ ID NO: 23,

(vii) VH: CDR1: positions 45-53 of SEQ ID NO: 19, CDR2: positions 71-78 of SEQ ID NO: 19, CDR3: positions 117-128 of SEQ ID NO: 19, VL: CDR1: SEQ ID NO: positions 47-58 of 26, CDR2: positions 76-78 of SEQ ID NO: 26, CDR3: positions 115-123 of SEQ ID NO: 26,

(viii) VH: CDR1: positions 45-53 of SEQ ID NO: 19, CDR2: positions 71-78 of SEQ ID NO: 19, CDR3: positions 117-128 of SEQ ID NO: 19, VL: CDR1: SEQ ID NO: positions 47-58 of 27, CDR2: positions 76-78 of SEQ ID NO: 27, CDR3: positions 115-123 of SEQ ID NO: 27, and

(ix) VH: CDR1: positions 45-53 of SEQ ID NO: 19, CDR2: positions 71-78 of SEQ ID NO: 19, CDR3: positions 117-128 of SEQ ID NO: 19, VL: CDR1: SEQ ID NO: positions 47-52 of 28, CDR2: positions 70-72 of SEQ ID NO: 28, CDR3: positions 109-117 of SEQ ID NO: 28.

In another preferred embodiment, the antibody capable of binding to CLDN18.2 preferably comprises one or more complementarity determining regions (CDRs), at least preferably a monoclonal antibody against CLDN18.2, preferably of the present invention. It is preferred that the monoclonal antibody against CLDN18.2 comprises a CDR3 variable region of the heavy chain variable region (VH) and/or light chain variable region (VL), preferably one or more complementarity determining regions (CDRs), At least preferably, the CDR3 of the heavy chain variable region (VH) and/or light chain variable region (VL) of a monoclonal antibody against CLDN18.2, preferably a monoclonal antibody against CLDN18.2 described herein. It is desirable to include a variable part. In one embodiment the one or more complementarity determining regions (CDRs) are selected from the sets of complementarity determining regions CDR1, CDR2 and CDR3 described herein. In a particularly preferred embodiment, the antibody having the ability to bind CLDN18.2 is preferably a monoclonal antibody against CLDN18.2, preferably the heavy chain variable of a monoclonal antibody against CLDN18.2 described herein. It is preferred that it comprises the complementarity determining regions CDR1, CDR2 and CDR3 of the region (VH) and/or the light chain variable region (VL), preferably the heavy chain variable regions (VH) and/or light chain variable regions ( It is preferable to include the complementarity determining regions CDR1, CDR2 and CDR3 of VL).

In one embodiment, an antibody comprising one or more CDRs, a set of CDRs, or a combination of sets of CDRs as described herein includes the CDRs and their intervening framework portions together. Preferably, the portion also comprises at least about 50% of either or both the first and fourth framework portions, wherein 50% is comprised of the C-terminal 50% of the first framework portion and the fourth framework portion. N-terminal 50%. The construction of antibodies made by recombinant DNA techniques can be accomplished by cloning, including introducing linkers to attach the variable regions of the invention to additional protein sequences, including immunoglobulin heavy chains, other variable regions (e.g., in diabody preparation), or protein labels. Alternatively, the N-terminal or C-terminal residues of the encoded variable region may be introduced by a linker introduced to facilitate other manipulation steps.

In one embodiment one or more CDRs, sets of CDRs or combinations of CDR sets described herein comprise the CDRs within a human antibody framework.

In the present invention, when an antibody contains a specific chain or a specific portion or sequence with respect to a heavy chain, preferably all heavy chains of the antibody include the chain, portion or sequence. This also applies to the light chain of an antibody.

As used herein, the term “nucleic acid” is intended to encompass DNA and RNA. Nucleic acids can be single-stranded or double-stranded, but preferably double-stranded DNA is preferred.

In the present invention, the term “expression” is mostly used in its general sense and includes the production of RNA or RNA and proteins/peptides.

This also includes partial expression of nucleic acids. Additionally, expression may be performed transiently or stably.

The teachings of the present invention for specific amino acid sequences, such as sequences appearing in a sequence listing, are directed to sequences functionally equivalent to the specific sequence, such as sequences that result in an amino acid sequence exhibiting properties equivalent to the specific amino acid sequence. It is understood that this also applies to variants of . One important property is maintaining the binding of the antibody to the target or maintaining the effector function of the antibody. Preferably, a sequence that is a variant relative to a specific sequence, when substituted for that specific sequence in the antibody, will preferably maintain binding of the antibody to CLDN18.2 and function as described in the present invention. , it is desirable to maintain functions such as CDC-mediated lysis or ADCC-mediated lysis.

Those skilled in the art will understand that, in particular, the CDR, hypervariable region and variable region sequences may be modified without losing the ability to bind to CLDN18.2. For example, the CDR portions may be identical or highly homologous to portions of the antibodies specified herein. By “highly homologous” it is intended that there may be 1 to 5, preferably 1 to 4, such as 1 to 3 or 1 or 2 substitutions within the CDRs. Additionally, hypervariable regions and variable regions may be modified such that they are substantially homologous to portions of the antibodies specifically described herein.

For the purposes of the present invention, a “variant” of an amino acid sequence includes amino acid insertion variants, amino acid addition variants, amino acid deletion variants, and/or amino acid substitution variants. Amino acid-deficient variants, including absence of the N-terminus and/or C-terminus of the protein, are also called N-terminus and/or C-terminus truncation variants.

Amino acid insertion variants include the insertion of one or two or more amino acids into a specific amino acid sequence. For amino acid sequence variants with insertions, one or more amino acid residues are inserted into specific regions of the amino acid sequence, although random insertions are also possible with appropriate screening of the products.

Amino acid addition variants include amino- and/or carboxy-terminal fusions of one or more amino acids, such as 1, 2, 3, 5, 10, 20, 30, 50 or more amino acids.

Amino acid deletion variants are characterized by the removal of one or more amino acids from the sequence, such as by removing 1, 2, 3, 5, 10, 20, 30, 50 or more amino acids. The absence may be in any region of the protein.

Amino acid substitution variants are characterized by removing at least one residue in the sequence and inserting another residue in its place. Modifications at regions of the amino acid sequence that are not conserved between homologous proteins or peptides and/or substitution of amino acids by others with similar properties are preferred. Preferably the amino acid changes in the protein variant are conservative amino acid changes, i.e. substitutions of similarly charged or uncharged amino acids. A conservative amino acid change involves the substitution of one member of the amino acid family related to its side chain. Naturally occurring amino acids are generally divided into four families: acidic amino acids (aspart, glutamate), basic amino acids (lysine, arginine, histidine), and nonpolar amino acids (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar amino acids (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). Phenylalanine, tryptophan, and tyrosine are sometimes classified together as aromatic amino acids.

Preferably, the degree of similarity, preferably the identity between a given amino acid sequence and an amino acid sequence that is a variant of the given amino acid sequence is at least about 60%, 65%, 70%, 80%, 81%, 82%, 83%, 84%. , 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. . The degree of similarity or identity is preferably at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, It is given for the amino acid region that is at least about 80%, at least about 90%, or about 100%. For example, if the reference amino acid sequence consists of 200 amino acids, the degree of similarity or identity is preferably at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, or at least about 140. , is given for at least about 160, at least about 180, or about 200 amino acids, preferably for consecutive amino acids. In a preferred embodiment, the degree of similarity or identity is given over the entire length of a reference amino acid sequence, such as the amino acid sequence appearing in the sequence listing. Alignment for sequence similarity determination, preferably sequence identification, can be performed using tools known in the art, preferably using the best sequence alignment, for example using Align, using standard settings, preferably EMBOSS::Needle, Matrix: Can be performed using Blosum62, Gap Open 10.0, Gap Extend 0.5.

“Sequence similarity” refers to the percentage of amino acids that are identical or exhibit conservative amino acid substitutions. “Sequence identity” between two amino acid sequences refers to the percentage of amino acids that are identical between the sequences.

The term "percentage identity" is intended to indicate the percentage of amino acid residues that are identical between the two sequences being compared, obtained after the best alignment, as such percentages are only statistical percentages and do not represent the randomly distributed differences between the two sequences. and the difference between the two sequences over the full length sequence. Sequence comparisons between two amino acid sequences are typically performed by optimally aligning the sequences described above and then comparing them, the comparison being performed by segmentation or "window of comparison" to identify local regions of sequence similarity. It's about sympathizing and comparing. In addition to manual alignment, optimal alignment between sequences for comparison can be accomplished using methods such as Smith and Waterman, 1981, Ads App. Math. 2, Local homology algorithm of 482, Neddleman and Wunsch, 1970, J. Mol. Biol. Local homology algorithm for 48, 443, Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 85, 2444 similarity search method, or computer programs using the above-described algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis. ) can be obtained by.

Percent identity is calculated by determining the number of identical positions between the two sequences being compared, dividing the number of identical positions by the number of positions compared, and multiplying the results obtained by 100 to determine the number of identical positions between the two sequences described above. Obtain the percent identity between them.

The term “transgenic animal” includes one or more transgenes, preferably antibody heavy and/or light chain transgenes, or transchromosomes (integrated or not integrated into the animal's native genomic DNA). It refers to an animal that has a genome that is capable of expressing the transgenes. For example, a transgenic mouse may carry either a human light chain transgene and a human heavy chain transgene or a human heavy chain transchromosome, such that the mouse contains CLDN18.2 antigen and/or cells expressing CLDN18.2. The mice produce human anti-CLDN18.2 antibodies when immunized with. The human heavy chain transgene can be integrated into the chromosomal DNA of a transgenic mouse, such as a HuMAb mouse such as HCo7 or HCol2 mice, or as in the case of a human heavy chain transgene, or the human heavy chain transgene can be transgeneized as described in WO 02/43478. In the case of chromosomal mice (eg, KM), it may be maintained extrachromosomally. Such transgenic and transchromosomal mice will be capable of producing multiple isotypes (e.g., IgG, IgA and/or IgE) of human monoclonal antibodies to CLDN18.2 through V-D-J recombination and isotype switching.

In the present invention, the meaning of "reduce", "reduce", and "inhibit" means reducing the proliferation level or expression level of cells to a certain level, preferably by 5% or more, 10% or more, or 20% or more. means the ability to reduce or cause an overall reduction of at least 50% and, best, at least 75%.

The term “increase” or “increase” refers to an increase, preferably by about at least 10%, preferably by at least 20%, preferably by at least 30%, more preferably by at least 40%, even more preferably by at least 50%, Even more preferably it relates to increasing or increasing by at least 80%, and most preferably by at least 100%, at least 200%, at least 500%, at least 1000%, at least 10000% or more.

Mechanism of mAb action

Although the following discussion provides consideration of the mechanisms underlying the therapeutic efficacy of the antibodies of the invention, it is not intended to limit the invention in any way.

The antibodies disclosed herein preferably interact with factors of the immune system, preferably via ADCC or CDC. Antibodies of the invention can also be used with targeting payloads (e.g., radioisotopes, drugs or toxins) that directly kill tumor cells, or have anti-tumor properties that may be compromised due to chemotherapeutic cytotoxic side effects in T lymphocytes. It can be used synergistically with traditional chemotherapy agents that attack tumors through complementary mechanisms of action that may involve an immune response. However, the antibodies of the invention may also exert their efficacy simply by binding to CLDN18.2 on the cell surface, thereby blocking proliferation of the cells.

Antibody Dependent Cell-Mediated Cytotoxicity

ADCC describes the cell killing capacity of effector cells, especially lymphocytes, as disclosed herein, which preferably requires target cells to be marked by antibodies.

ADCC occurs when antibodies bind antigens, preferably on tumor cells, and antibody Fc domains engage Fc receptors on the surface of immune effector cells. Several families of Fc receptors have been identified, and specific cell populations express characteristic Fc receptors. ADCC can be considered a mechanism to directly induce varying degrees of immediate tumor destruction, resulting in antigen presentation and induction of tumor-specific T cell responses. Preferably, in vivo induction of ADCC results in tumor-specific T cell responses and host-derived antibody responses.

Complement-dependent cytotoxicity

CDC is another method of cell killing that can be directed by antibodies. IgM is the most effective isotype for complement activation. IgG1 and IgG3 are very effective in directing CDC through the classical complement activation pathway. Preferably, in this cascade, the formation of antigen-antibody complexes brings antibody molecules, including IgG molecules, into close proximity on the CH2 domain, resulting in the uncloaking of multiple C1q binding sites (C1q is a complement binding site). It is one of the three subfactors of C1). Ideally, these exposed C1q binding sites convert the previously low affinity C1q-IgG interaction to high avidity, which sets off a cascade involving a series of other complement proteins and effectors. Resulting in proteolytic release of cell chemotactic/activators C3a and C5a. Preferably, at the end of the membrane formation, the complement cascade attacks the complex, which forms a pore in the cell membrane that facilitates free passage of solutes and water into and out of the cell.

Antibodies of the invention can be produced by a variety of techniques, including conventional monoclonal antibody methodology, such as the standard somatic cell hybridization technique of Kohler and Milstein, Nature 256: 495 (1975). Although somatic cell hybridization procedures are preferred, other techniques for producing monoclonal antibodies can be deployed, such as viral or tumor transformation of B-lymphocytes or phages, which represent techniques using libraries of antibody genes.

The preferred animal system for producing hybridomas secreting monoclonal antibodies is the murine lineage. Hybridoma production in mice is a very well-established procedure. Immunization protocols and techniques for isolation of immunized splenocytes for fusion are well known in the art. Fusion procedures with fusion partners (e.g., murine myeloma cells) are also known.

Other preferred animal systems for producing hybridomas secreting monoclonal antibodies are the rat and rabbit systems (e.g., Spieker-Polet et al., Proc. Natl. Acad. Sci. U.S.A. 92:9348 (1995) , Rossi et al., published in Am. J. Clin. Pathol. 124: 295 (2005).

In another preferred embodiment, human monoclonal antibodies can be generated using transgenic or transchromosomal mice that carry out parts of the human immune system rather than the mouse system. Such transgenic and transchromosomal mice include those known as HuMab mice and KM mice, respectively, and are collectively referred to herein as “transgenic mice.” Production of human antibodies in such transgenic mice can be performed as detailed for CD20 in WO2004 035607.

Another strategy for generating monoclonal antibodies is described in, e.g., Babcock et al., 1996; Direct isolation of genes encoding antibodies from lymphocytes producing antibodies of defined specificity, referring to a novel strategy for producing monoclonal antibodies from single isolated lymphocytes producing antibodies of defined specificity. . For further details on recombinant antibody engineering, see also Welschof and Kraus, Recombinant antibodes for cancer therapy ISBN-0-89603-918-8 and Benny K.C. Lo Antibody Engineering ISBN 1-58829-092-1.

To produce an antibody, a carrier-conjugated peptide derived from the antigenic sequence, i.e. the sequence to which the antibody is to be directed, a recombinantly expressed enriched preparation of CLDN18.2 as described or a fragment thereof and/or CLDN18.2 or its Mice can be immunized with cells expressing the fragment. Alternatively, mice can be immunized with DNA encoding the antigen or fragment thereof. If immunization with a purified or enriched preparation of the antigen does not produce antibodies, mice can also be immunized with cells expressing the antigen, such as a cell line, to stimulate an immune response.

Immune responses can be monitored over the course of the immunization protocol with plasma and serum samples obtained by tail vein or retroorbital bleeds. Mice with sufficient titers of immunoglobulin can be used for fusion. Mice can be sacrificed and boosted intraperitoneally or intravenously with antigen-expressing cells 3 days prior to spleen removal to increase the proportion of specific antibody-secreting hybridomas.

To generate hybridomas that produce monoclonal antibodies, lymph node cells and spleen cells from immunized mice can be isolated and fused to an appropriate immortalized cell line, including a mouse myeloma cell line. The resulting hybridomas can then be screened for production of antigen-specific antibodies. Each well can then be screened by ELISA for antibody secreting hybridomas. Antibodies specific to the antigen can be identified by immunofluorescence and FACS using antigen-expressing cells. Antibody-secreting hybridomas can be replated, screened again, and subcloned by limiting dilution if still positive for monoclonal antibodies. Stable subclones can then be cultured in vitro to produce antibodies in tissue culture medium for characterization.

Antibodies can also be produced in host cell transfectomas, for example, using a combination of recombinant DNA technology and gene transfection methods well known in the art (Morrison, S. (1985) Science 229: 1202).

For example, in one embodiment, the gene(s) of interest, e.g., antibody genes, may be expressed by the GS gene expression system disclosed in WO 87/04462, WO 89/01036 and EP 338 841 or other expression systems well known in the art. It can be ligated into expression vectors, including eukaryotic expression plasmids. Purified plasmids with cloned antibody genes can be introduced into other eukaryotic cells such as CHO cells, NS/0 cells, HEK293T cells or HEK293 cells or alternatively plant derived cells, mold or yeast cells. You can. The method used to introduce these genes may be methods described in the art, including electroporation, lipofectin, lipofectamine or others. After introduction of these antibody genes within a host cell, cells expressing the antibody are identified and selected. These cells are then amplified for their expression level and represent transfectomas that can be upscaled to produce antibodies. Recombinant antibodies can be isolated and purified from these culture supernatants and/or cells.

Alternatively, cloned antibody genes can be expressed in other expression systems, including prokaryotic cells, including microorganisms, including E. coli. Antibodies can also be produced in transgenic non-human animals, such as in milk from sheep or rabbits or in hen eggs, or in transgenic plants (Verma, R., et al. (1998) J. Immunol. Meth. 216: 165-181; Pollock, et al. (1999) J. Immunol. Meth. 231: 147-157; and Fischer, R., et al. (1999) Biol. Chem. 380: 825- 839 reference).

Chimerization

Murine monoclonal antibodies can be used as therapeutic antibodies in humans when labeled with a toxin or radioisotope. Unlabeled murine antibodies are highly immunogenic in humans when applied repeatedly, resulting in reduced therapeutic effectiveness. The immunogenicity of murine antibodies is mediated by the heavy chain constant region. The immunogenicity of murine antibodies in humans can be reduced or completely prevented if the individual antibodies are chimerized or humanized. Chimeric antibodies are antibodies with different portions derived from different animal species, including those with variable regions derived from murine antibodies and human immunoglobulin constant regions. Chimerization of antibodies is accomplished by combining the constant regions of human heavy and light chains with the variable regions of murine antibody heavy and light chains (e.g., Kraus et al., in Methods in Molecular Biology series, Recombinant antibodies for cancer therapy ISBN-0- initiated by 89603-918-8). In a preferred manner, chimeric antibodies can be generated by linking the constant region of a human kappa light chain to the variable region of a murine light chain. Another preferred method of antibody chimerization involves linking the constant region of a human lambda light chain to the variable region of a murine light chain. Preferred heavy chain constant regions for generating chimeric antibodies include IgG1, IgG3, and IgG4. Other preferred heavy chain constant regions for the generation of chimeric antibodies include IgG2, IgA, IgD, and IgM.

Humanization

*Antibodies react with target antigens primarily through amino acid residues present in the six heavy and light chain CDRs. For this reason, amino acid sequences within the CDR appear more diverse among each antibody than sequences outside the CDR. Since CDR sequences play a critical role in most antibody-antigen reactions, CDR sequences containing CDR sequences from specifically occurring antibodies are grafted onto framework sequences from different antibodies with different properties. By constructing expression vectors, it is possible to express recombinant antibodies that mimic the properties of specific naturally occurring antibodies (e.g., Riechmann, L. et al. (1998) Nature 332: 323-327; Jones, P. et al. (1986) Nature 321: 522-525; and Queen, C. et al. (1989) Proc. Natl. Acad. Sci. U.S. A. 86: 10029-10033). Such framework sequences can be obtained from public DNA databases containing germline antibody gene sequences. These germline sequences will differ from the mature antibody gene sequences because they will not contain the fully integrated variable genes generated by the binding of V(D)J during the maturation phase of the B cell. Germline gene sequences will also differ from each high affinity secondary repertoire antibody evenly distributed throughout the variable region.

The ability of an antibody to bind an antigen can be detected using standard binding assays (e.g., ELISA, Western blot, immunofluorescence, and flow cytometry).

To purify antibodies, selected hybridomas can be grown in 2-liter spinner-flasks for monoclonal antibody purification. Alternatively, antibodies can be produced in a dialysis-based bioreactor. The supernatant can be filtered and, if necessary, concentrated before affinity chromatography with Protein G-Sepharose or Protein A-Sepharose. Eluted IgG can be checked by gel electrophoresis and high-performance liquid chromatography to ensure purity. The buffer solution can be replaced with PBS and the concentration is measured by OD280 using an extinction coefficient of 1.43. Monoclonal antibodies can be aliquoted and stored at -80°C.

To determine if selected monoclonal antibodies bind to a unique epitope, site-directed or multi-site directed mutagenesis can be used.

To determine the isotype of the antibodies, isotype ELISA from various commercial kits (e.g., Zymed, Roche Diagnostics) was performed. Wells of a microtiter plate can be coated with anti-mouse Ig. After blocking, plates were reacted with monoclonal antibodies or purified isotype controls at ambient temperature for 2 hours. The wells can then be reacted with mouse IgG1, IgG2a, IgG2b or IgG3, IgA or mouse IgM-specific peroxidase-conjugated probes. After washing, the plates can be developed with ABTS substrate (1 mg/ml) and analyzed at 405-650 OD. Alternatively, the IsoStrip Mouse Monoclonal Antibody Isotyping Kit (Roche, Cat. No. 1493027) can be used as described by the manufacturer.

Flow cytometry was used to demonstrate the binding of monoclonal antibodies to live cells expressing the antigen or the presence of antibodies in the serum of immunized mice. Cell lines expressing the antigen natively or after transfection and negative controls lacking antigen expression (grown under standard growth conditions) were mixed with hybridoma supernatants or various concentrations of monoclonal antibodies in PBS containing 1% FBS. and can be incubated at 4°C for 30 minutes. After washing, the APC- or Alexa647-labeled anti-IgG antibody can bind to the antigen-bound monoclonal antibody under the same conditions as the initial antibody staining. Samples can be analyzed by flow cytometry with a FACS instrument using light and side scatter properties to gate single, viable cells. To distinguish antigen-specific monoclonal antibodies from non-specific binders in a single measurement, co-transfection methods can be implemented. Cells transiently transfected with a plasmid encoding the antigen and a fluorescent marker can be stained as described above. Transfected cells can be detected in a different fluorescence channel than antibody-stained cells. As the majority of transfected cells express both transgenes, antigen-specific monoclonal antibodies bind preferentially to cells expressing the fluorescent marker, whereas non-specific antibodies bind at comparable rates to untransfected cells. Combine with Alternative assays using fluorescence microscopy can be used in addition to or instead of flow cytometry assays. Cells can be stained exactly as described above and examined by fluorescence microscopy.

Immunofluorescence microscopy can be used to demonstrate the presence of antibodies in the serum of immunized mice or the binding of monoclonal antibodies to living cells expressing the antigen. For example, cell lines expressing antigen spontaneously or after transfection or negative controls lacking antigen expression were cultured in DMEM supplemented with 10% FCS, 2 mM L-glutamine, 100 IU/ml penicillin, and 100 μg/ml streptomycin. Cultured on chamber slides under standard growth conditions in F12 medium. Cells are then fixed with methanol or paraformaldehyde or left untreated. It is then reacted with a monoclonal antibody against the antigen at 25°C for 30 minutes. After washing, these cells were reacted with Alexa555-labeled anti-mouse IgG secondary antibody (Molecular Probes) under the same conditions. It can then be examined under a fluorescence microscope.

Cell extracts from cells expressing the antigen or appropriate negative controls can be prepared and subjected to SDS polyacrylamide gel electrophoresis. After electrophoresis, the separated antigen is transferred to a nitrocellulose membrane, blocked, and probed with the monoclonal antibody to be tested. IgG binding is detected using anti-mouse IgG peroxidase and developed with ECL substrate.

Antibodies can be obtained by immunohistochemistry in a manner well known to those skilled in the art, e.g., from cancer-free mice obtained from patients during surgery or from mice bearing xenografted tumors inoculated with cell lines expressing the antigen, either spontaneously or after transfection. Reactivity to an antigen can be additionally measured from tissue or cancer tissue samples by using paraformaldehyde or acetone fixed cryosections, or paraffin-embedded and paraformaldehyde fixed tissue sections. For immunostaining, antibodies reactive to the antigen are incubated and then horseradish-peroxidase conjugated with goat anti-mouse or goat anti-rabbit antibodies (DAKO) according to the distributor's instructions.

Antibodies can be tested for their ability to mediate phagocytosis and kill cells expressing CLDN18.2. Initial screening can be accomplished by testing monoclonal antibody activity in vitro, prior to testing in in vivo models.

Antibody Dependent Cell-Mediated Cytotoxicity (ADCC):

Briefly, polymorphonuclear cells (PMNs), NK cells, monocytes, monocytes, or other effector cells from healthy donors can be purified by Ficoll Hypark density centrifugation and then contaminating red blood cells can be lysed. Washed effector cells were suspended in RPMI supplemented with 10% heat-inactivated FCS or, alternatively, 5% heat-inactivated human serum and incubated with ⁵¹ Cr labeled target cells expressing CLDN18.2. Mix at various ratios of effector cells to target cells. Alternatively, target cells can be labeled with a fluorescence enhancing ligand (BATDA). Highly fluorescent chelation of europium with the above-mentioned enhancing ligands from dead cells can be measured fluorimetrically. Another alternative technique may be to use transfection of target cells with luciferase. Lucifer yellow added in this way can only be oxidized by living cells. Purified anti-CLDN18.2 IgGs can then be added at various concentrations. Irrelevant human IgG can be used as a negative control. Depending on the type of effector cell used, the analysis can be performed at 37°C for as little as 4 hours and as much as 20 hours. Samples are analyzed for cell lysis by measuring ⁵¹ Cr excretion or the presence of EuTDA chelating compounds in the culture supernatant. Alternatively, luminescence due to oxidation of lucifer yellow can be a measure of living cells.

Anti-CLDN18.2 monoclonal antibodies can also be tested in various combinations to determine whether cell lysis increases with multiple monoclonal antibodies.

Complement Dependent Cytotoxicity (CDC)

The CDC mediating ability of monoclonal anti-CLDN18.2 antibodies can be tested using a variety of known techniques. For example, serum for complement can be obtained from blood according to methods known to those skilled in the art. Different methods are used to measure the CDC activity of mAbs. For example, ⁵¹ Cr excretion can be measured or elevated cell membrane permeability can be measured using a PI exclusion assay. Briefly, target cells can be washed and incubated at 5 x 10 ⁵ /ml with various concentrations of mAb for 10-30 minutes at 37°C or room temperature. Then, add serum or plasma until the final concentration reaches 20% (v/v) and leave the cells at 37°C for 20-30 minutes. All cells from each sample are added to the PI solution in the FACS tube. This mixture can be immediately analyzed by flow cytometric analysis using a FACS array.

In another assay, CDC induction is dependent on adherent cells. In one embodiment of this assay, cells are seeded at a density of 3 x 10 ⁴ /well in tissue culture flat bottom microtiter plates 24 hours prior to analysis. The next day, the growth medium is removed and the cells are incubated in triplicate with antibodies. Control cells were incubated with growth medium or growth medium containing 0.2% saponin to determine background or maximum lysis, respectively. After incubation at room temperature for 20 minutes, the supernatant is removed, 20% (v/v) human plasma or serum in DMEM (preheated at 37°C) is added to the cells, and incubated at 37°C for another 20 minutes. All cells from each sample were added to propidium iodide solution (10 μg/ml). Then, the supernatant is replaced with PBS containing 2.5 μg/ml ethidium bromide and fluorescence emission upon excitation at 520 nm is measured at 600 nm using a Tecan Safire. The percentage of specific lysis is calculated as follows: % specific lysis = (fluorescent sample-fluorescent background)/(fluorescent maximum lysis-fluorescent background) x 100

Induction of apoptosis and inhibition of cell proliferation by monoclonal antibodies:

To test the ability to initiate apoptosis, for example, a monoclonal anti-CLDN18.2 antibody is transfected into CLDN18.2 positive tumor cells, such as SNU-16, DAN-G, KATO-III or CLDN18.2. Incubation with infected tumor cells can be done at 37°C for approximately 20 hours. After these cells are collected, they are washed in Annexin-V conjugated buffer (BD Life Sciences) and incubated with Annexin V conjugated to FITC or APC (BD Life Sciences) in the dark for 15 minutes. All cells from each sample were added to PI solution (10 μg/ml in PBS) in a FACS tube and immediately evaluated by flow cytometry (as described above). Alternatively, general inhibition of cell proliferation by monoclonal antibodies can be detected by commercial kits. The DELFIA Cell Proliferation Kit (Perkin-Elmer, Cat. No. AD0200) is a non-isotope immunosorbent assay based on measurement of 5-bromo-2'-deoxyuridine (BrdU) incorporation during DNA synthesis in proliferating cells in microplates. It is an analysis method. Mixed BrdU is detected using europium-labeled monoclonal antibodies. To enable antibody detection, cells are fixed and DNA is denatured using Fix solution. Here, DELFIA inducer is added to the solution to wash away unbound antibodies and separate europium ions from labeled antibodies. They form highly fluorescent chelating compounds with components of the DELFIA inducer. The measured fluorescence - detection using time-resolved fluorometry - is proportional to DNA synthesis in the cells of each well.

preclinical studies

To determine efficacy in controlling the growth of tumor cells expressing CLDN18.2, monoclonal antibodies that bind to CLDN18.2 were tested in an in vivo model (e.g., SNU-16, DAN-G, KATO-III, or After transfection, experiments can be performed in immunodeficient mice bearing xenografted tumors, inoculated with a cell line expressing CLDN18.2, such as HEK293.

In vivo studies following injection of CLDN18.2-expressing positive cells into immunocompromised mice or other animals can be performed using the antibodies of the present invention. To measure the effectiveness of antibodies in preventing tumor formation or tumor-related symptoms, antibodies can be administered to tumor-free mice followed by tumor cells. Antibodies can be administered to tumor-bearing mice to determine the therapeutic efficacy of each antibody to reduce tumor growth, metastasis, or tumor-related symptoms. Antibody application can measure the synergistic effect or potential toxicity of combinations of other antibodies and other agents such as cystostatic drugs, growth factor inhibitors, cell cycle blockers, and angiogenesis inhibitors. To analyze the toxic side effects caused by antibodies, animals can be injected with antibodies or control reagents and closely examined for symptoms that may be related to CLDN18.2 antibody treatment. Possible side effects of in vivo application of CLDN18.2 antibodies include toxicity in CLDN18.2 expressing tissues, including the stomach. Antibodies that recognize CLDN18.2 in humans or other species such as mice are particularly useful for predicting potential side effects resulting from the application of monoclonal CLDN18.2 antibodies in humans.

Mapping of epitopes recognized by antibodies is described in "Epitope Mapping Protocols (Methods in Molecular Biology) by Glenn E. Morris ISBN-089603-375-9 and in 'Epitope Mapping: A Practical Approach" Practical Approach Series, 248 by Olwyn M. R. Westwood, This can be done as detailed in Frank C. Hay.

The compounds and agents described in the present invention may be administered in the form of appropriate pharmaceutical compositions.

Pharmaceutical compositions are usually presented in unit dosage form and can be prepared in a known manner. The pharmaceutical composition may, for example, be in the form of a solution or suspension.

The pharmaceutical composition may contain salts, buffering agents, preservatives, carriers, diluents and/or excipients, all of which are preferably pharmaceutically acceptable. “Pharmaceutically acceptable” refers to the non-toxicity of an agent that does not interact with the action of the active ingredients of the pharmaceutical composition.

Pharmaceutically acceptable salts can be prepared using pharmaceutically unacceptable salts, and these also belong to the present invention. Pharmaceutically acceptable salts of this type include, but are not limited to, those prepared from the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, citric acid, formic acid, malonic acid, Succinic acid, etc. Pharmaceutically acceptable salts may also be prepared as alkali metal salts or alkaline earth metal salts, such as sodium, potassium or calcium salts.

Buffering agents suitable for use in pharmaceutical compositions include acetates, citrates, borates and phosphates.

Preservatives suitable for use in pharmaceutical compositions include benzalkonium chloride, chlorobutanol, parabens and thimerosal.

Injectable formulations may contain pharmaceutically acceptable excipients such as Ringer Lactate.

The term “carrier” refers to an organic or inorganic ingredient, natural or synthetic, with which an active ingredient is combined to facilitate, enhance or enable the application of the active ingredient. According to the present invention, “carrier” also includes one or more compatible solid or liquid fillers, diluents or encapsulating agents suitable for administration to a patient.

Possible carrier agents for parenteral administration include, for example, sterile water, Ringer, Ringer lactate, sterile sodium chloride solution, polyalkylene glycols, hydrogenated naphthalenes, especially biocompatible lactide polymers, lactide/glycolide copolymers or poly It is an oxyethylene/polyoxypropylene copolymer.

As used herein, the term "excipient" refers to any agent that may be present in a pharmaceutical composition but is not an active ingredient, such as a carrier, binder, lubricant, thickening agent, surfactant, preservative, emulsifier, buffer, flavoring agent or colorant. It is intended to be.

The agents and compositions described herein may be administered via any conventional route, such as parenteral administration, including injection or infusion. Administration may preferably be by parenteral administration, including, for example, intravenously, intraarterially, subcutaneously, intradermally, or intramuscularly.

Compositions suitable for parenteral administration generally include sterile aqueous or non-aqueous formulations of the active compound, which are preferably isotonic with the blood of the recipient. Suitable carriers and solvents are Ringer's solution and isotonic sodium chloride solution. In addition, usually sterile fixed oils are used as solution or suspension medium.

The agents and compositions described herein are administered in effective amounts. “Effective amount” is the amount that, alone or in combination with additional doses, produces the desired response or desired effect. When treating a particular disease or condition, the desired response preferably relates to inhibiting the course of the disease. This includes slowing the progression of the disease, particularly preventing or reversing the progression of the disease. The desired response in treating a disease or condition may also be to slow or prevent the onset of the disease or condition.

The effective amount of an agent or composition described herein may vary depending on the condition being treated, the severity of the disease, the individual variables of the patient, such as age, physiological state, size and weight, duration of treatment, type of concomitant therapy (if any), and the specific administration. Depends on the route and similar factors. Accordingly, the dosage of the agents described herein will vary depending on these various variables. If the patient's response is insufficient with the initial dose, higher doses (or effectively higher doses achieved by other, more local, routes of administration) may be used.

The agents and compositions described herein can be administered to patients, e.g., in vivo, to treat or prevent a variety of diseases such as those described herein. Preferred patients include human patients suffering from a disease that can be corrected or alleviated by administration of the agents and compositions described herein. These include diseases involving cells characterized by altered expression patterns of CLDN18.2.

For example, in one embodiment, the antibodies described herein can be used to treat patients suffering from a cancerous disease, e.g., a cancerous disease such as that described herein characterized by the presence of cancer cells expressing CLDN18.2. there is.

The pharmaceutical composition and treatment method according to the present invention can also be used for immunization or vaccination for the prevention of the diseases described in the present invention.

Next, the present invention will be described in more detail with reference to examples, but the scope of the present invention is not limited to these.

Example

Example 1: Human gastric cancer cell lines CLDN18 .2 Expression using chemotherapy agents in vitro stabilized by treatment

KatoIII cells, a human gastric tumor cell line, were cultured in RPMI 1640 medium (Invitrogen) containing 20% FCS (Perbio) and 2 mM Glutamax (Invitrogen) at 37°C and 5% CO ₂ in the presence or absence of cytostatic compounds. . Epirubicin (Pfizer) at a concentration of 10 or 100 ng/ml, 5-FU (Neofluor from NeoCorp AG) at a concentration of 10 or 100 ng/ml, and oxaliplatin (Hospira) at a concentration of 50 or 500 ng/ml. Concentration was tested. A mixture of these three compounds (EOF; epirubicin 10 ng/ml, oxaliplatin 500 ng/ml, 5-FU 10 ng/ml) was also tested. Cells were released from cell cycle arrest by culturing 8x10 ⁵ KatoIII cells in a 6-well tissue culture plate at 37°C and 5% CO ₂ for 96 hours without changing the medium, or by culturing them for 72 hours and then culturing them in standard medium for 24 hours. Cells were harvested, washed and analyzed using EDTA/trypsin.

For extracellular detection of CLDN18.2, cells were stained with the monoclonal anti-CLDN18.2 antibody IMAB362 (Ganymed) or an isotype-matched control antibody (Ganymed). As a second reagent, chlorine-anti-huIgG-APC from Dianova was used.

Cell cycle stages were determined based on measurements of cellular DNA content. This process allows cells in the G1-, S-, or G2-phase of the cell cycle to be distinguished from each other. In S-phase, DNA replication occurs, while in G2-phase, cells grow and prepare for mitosis. Cell cycle analysis was performed using BD Biosciences' CycleTEST PLUS DNA Reagent Kit according to the manufacturer's protocol. Flow cytometry data collection and analysis were performed using BD FACS CantoII (BD Biosciences) and FlowJo (Tree Star) software.

Columns in Figure 1A and B represent the percentage of cells in G1-, S-, or G2-phase of the cell cycle. KatoIII cells cultured with medium showed cell cycle arrest mainly in G1-phase. Cells treated with 5-FU mainly stayed in S-phase. Epirubicin- or EOF-treated KatoIII cells were mainly arrested in the cell cycle in G2-phase. KatoIII cells treated with oxaliplatin mainly appeared in G1- and G2-phase. As shown in Figure 1C, cell cycle arrest in S-phase or G2-phase results in stabilization or upregulation of CLDN18.2. Once cells exit any phase of the cell cycle (Figure 1B), CLDN18.2 expression on the cell surface of KatoIII cells is upregulated (Figure 1D).

NUGC-4 and KATO III cells were incubated with 5-FU + OX (10 ng/ml 5-FU and 500 ng/ml oxaliplatin), EOF (10 ng/ml epirubicin, 500 ng/ml oxaliplatin and 10 ng/ml 5 -FU) or FLO (10 ng/ml 5-FU, 50 ng/ml folinic acid and 500 ng/ml oxaliplatin) for 96 hours. RNA from chemotherapy-pretreated NUGC-4 and KATO III cells was isolated and converted to cDNA. CLDN18.2 transcript levels were analyzed by quantitative real-time PCR. The results are shown in Figure 2a as relative expression values compared to the former level of the housekeeping gene HPRT. Figure 2b is a diagram showing Western blot of actin loading control and CLDN18.2 of untreated and treated NUGC-4 cells. Luminescent signal intensity was expressed as a percentage relative to actin.

Pretreatment of NUGC-4 and KATO III cells with EOF, FLO and 5-FU + OX combination chemotherapy resulted in increased RNA and protein levels of CLDN18.2, as shown by quantitative real-time PCR (Figure 2a) and Western blot (Figure 2b). This has increased.

EOF (10 ng/ml epirubicin, 500 ng/ml oxaliplatin, and 10 ng/ml 5-FU) or FLO (10 ng/ml 5-FU, 50 ng/ml folinic acid, and 500 ng/ml 5-FU) for 96 h by flow cytometry. The binding of IMAB362 to NUGC-4 and KATO III gastric cancer cells treated with ng/ml oxaliplatin was analyzed. The amount of CLDN18.2 protein targetable by IMAB362 on the surface of gastric cancer cell lines increased as shown in Figure 2c. This effect was most pronounced in cells pretreated with EOF or FLO.

KatoIII cells were pretreated with irinotecan or docetaxel for 4 days and then analyzed for CLDN18.2 expression and cell cycle arrest. Treatment of cells with irinotecan resulted in inhibition of cell growth and cell cycle arrest in S/G2-phase in a dose-dependent manner (Figure 3). Treatment of cells with docetaxel resulted in inhibition of cell growth and cell cycle arrest in G2-phase in a dose-dependent manner (Figure 3).

Example 2: Human stomach cancer cells by chemotherapy Preprocessing is more Results in IMAB362-mediated ADCC with efficiency

10 ng/ml 5-FU and 500 ng/ml oxaliplatin (5-FU + OX), 10 ng/ml epirubicin, 500 ng/ml oxaliplatin and 10 ng/ml 5-FU (EOF) or 10 ng/ml NUGC-4 gastric cancer cells pretreated with 5-FU, 50 ng/ml folinic acid and 500 ng/ml oxaliplatin (FLO) for 96 h (effector:target ratio 40:1) or untreated were used as targets for IMAB362- Mediated ADCC was investigated. EC ₅₀ values were collected from 7 healthy donors for untreated cells and NUGC-4 cells pretreated with EOF, FLO or 5-FU + OX.

As shown in Figure 4A, the dose/response curve for pretreated cells shifted upward and to the left compared to untreated target cells. This resulted in higher maximal lysis and lower EC ₅₀ values by one-third compared to untreated cells (Figure 4b).

Peripheral blood mononuclear cells (PBMCs), including NK cells, monocytes, mononuclear cells, or other effector cells, obtained from healthy human donors were purified by Ficoll Hypaque density centrifugation. Washed effector cells were inoculated into X-Vivo medium. KatoIII cells, which endogenously express CLDN18.2 and originate from the stomach, were used as target cells in this setting. The target cells stably expressed lucifer yellow and luciferase, which are oxidized only by living cells. Purified anti-CLDN18.2 antibody IMAB362 was added at various concentrations, and an irrelevant chimeric huIgG1 antibody was used as an isotype control antibody. Samples were analyzed for cytolysis by measuring the amount of luminescence resulting from oxidation of lucifer yellow, a value indicative of the number of viable cells remaining after IMAB362 induced cytotoxicity. KatoIII pretreated for 3 days with irinotecan (1000 ng/ml), docetaxel (5 ng/ml) or cisplatin (2000 ng/ml) were compared to untreated medium cultured target cells and IMAB362 induced ADCC was quantified.

KatoIII cells pretreated with irinotecan, docetaxel, or cisplatin for 3 days showed lower viable cell levels compared to medium-cultured target cells (Figure 5a), and Claudin18.2 expression in cells pretreated with irinotecan, docetaxel, or cisplatin was significantly higher than medium-cultured target cells. increased compared to cells (Figure 5b).

Additionally, pretreatment of KatoIII cells with irinotecan, docetaxel or cisplatin enhanced the ability of IMAB362 to induce ADCC (Fig. 5c,d).

Example 3: Chemotherapy further increases the efficiency of IMAB362-induced CDC

The effect of chemotherapy agents on IMAB362-induced CDC was analyzed by pretreating KATO III gastric cancer cells with 10 ng/ml 5-FU and 500 ng/ml oxaliplatin (5-FU + OX) for 48 hours. A representative dose response curve of IMAB362-induced CDC using KATO III cells pretreated with chemotherapy agents is shown in Figure 6. Pretreatment of tumor cells for 48 hours increased the intensity of IMAB362 inducing CDC, resulting in a higher maximum cell lysis of pretreated tumor cells compared to untreated cells.

Example 4: IMAB362 - mediated ADCC performing immunity effector Cellular capacity is not weakened by chemotherapy treatment

Chemotherapeutic agents used in EOF or FLO therapy are very potent in inhibiting target cell proliferation. To investigate the side effects of chemotherapy on effector cells, PBMCs from healthy donors were incubated with 10 ng/ml epirubicin, 500 ng/ml oxaliplatin, and 10 ng/ml 5-FU (EOF) for 72 h prior to ADCC analysis. ) or treated with 10 ng/ml 5-FU, 50 ng/ml folinic acid, and 500 ng/ml oxaliplatin (FLO). Figure 7A shows EC ₅₀ values from four healthy donors and Figure 7B shows representative dose/response curves of ADCC induced using EOF or FLO pretreated effector cells. IMAB362-induced ADCC of NUGC-4 gastric carcinoma cells is not compromised by EOF or FLO chemotherapy.

Example 5: Z.A. /IL-2 treatment is performed on peripheral blood mononuclear cell( PBMC ) to the optimal level of culture confirm

The effect of ZA/IL-2 on the proliferation of PBMC cultures was evaluated in vitro . PBMCs were harvested from healthy human donors and cultures were treated with a single dose of ZA. IL-2 was added every 3-4 days. Specifically, PBMCs derived from three different healthy human donors (#1, #2, #3) were incubated with 1 μM ZA plus high (300 U/ml) or low (25 U/ml) doses of IL-2. 8A. PBMCs from the same donor were additionally cultured in RPMI medium (1x10 ⁶ cells/ml) for 14 days with 300 U/ml IL-2 plus ZA or without ZA. cf. Figure 8b. Increase in cell number was detected by counting viable cells on days 6, 8, 11 and 14.

In medium supplied with a high dose of IL-2, cells proliferated about 2-5 times more than in cultures supplied with a low dose of IL-2 (FIG. 8a). Cell proliferation in medium without ZA was approximately two-fold lower compared to cells grown in medium containing ZA (Figure 8b). These data show that it is necessary to apply both ZA and IL-2 compounds in combination to ensure proper proliferation of cells.

Example 6: Z.A. /IL-2 treatment is PBMC Among cultures Vγ9Vδ2 Results in massive proliferation of T-cells

PBMCs were cultured in RPMI medium supplemented with 300 U/ml IL-2 and with or without 1 μM ZA for 14 days. The percentage of Vγ9+Vδ2+ T cells in the CD3+ lymphocyte population (Figure 9A) and the percentage of CD16+ cells in the CD3+Vγ9+Vδ2+ T cell population (Figure 9B) were measured by multicolor FACS on days 0 and 14. Results were scored in a scatter plot for each donor. Figure 9c is a scatter plot showing the temporal increase (enrichment) in the number of CD3+ Vγ9+Vδ2+ and CD3+CD16+ Vγ9+Vδ2+ T cells in the lymphocyte population. The amount of cells inoculated on day 0 and the amount of cells harvested on day 14 were considered.

The addition of IL-2 in PBMC cultures is required for lymphocyte survival and growth. They proliferate effectively in cultures supplemented with 300 U/ml IL-2. FACS analysis using Vγ9 and Vδ2 specific antibodies showed that ZA/IL-2 specifically induced the accumulation of Vγ9Vδ2 T cells (FIG. 9a). After 14 days, the CD3+ lymphocyte population contained up to 80% Vγ9Vδ2 T cells. While a fraction of Vγ9Vδ2 T cells expressed CD16, the degree of enrichment of these cells in the CD3+ lymphocyte population was 10-700-fold, depending on the donor (Figures 9b and 9c). CD16+Vγ9+Vδ2+ T cells in cultures are 10-600-fold higher compared to cultures grown without ZA (Figure 9C). We concluded that ZA/IL-2 treatment of PBMCs in vitro resulted in upregulation of the ADCC-mediated FcγIII receptor CD16 in a significant proportion of γδ T cells.

Example 7: IL-2 in a dose-dependent manner Vγ9Vδ2 Affects proliferation of T cells

The addition of ZA to the culture is the most important factor in inducing the development of Vγ9Vδ2 T cells. It is well known that IL-2 is required for T cell growth and survival.

PBMCs were cultured in RPMI medium supplemented with 1 μM ZA for 14 days with increasing concentrations of IL-2. IL-2 was added on days 0 and 4. Enrichment of CD16+Vγ9+Vδ2+ T cells in the CD3+ lymphocyte population was detected by multicolor FACS staining on days 0 and 14. To compare different donors with each other, the amount of CD16+Vγ9+Vδ2+ T cells harvested after incubation with 600 U/ml IL-2 was taken as 100%; cf. Figure 10, left. Additionally, the ADCC activity of isolated cultures grown for 14 days with increasing concentrations of IL-2 was examined; cf. Figure 10, right.

The present inventors confirmed by dose response curve that IL-2 also promoted the growth and survival of the Vγ9Vδ2 T cell subset. By adding low concentrations of IL-2 to the medium, a correlation was found between IL-2 dose and the percentage of CD16+Vγ9Vδ2 T cells in the CD3+ lymphocyte population (Figure 10, left). The ADCC activity of cells grown at higher concentrations of IL-2 (150-600U/ml) was further improved compared to cells grown at low concentrations of IL-2 (Figure 10, right).

Example 8: ZA is In monocytes and cancer cells IPP induces creation and both Vγ9Vδ2 Promotes proliferation of T cells

Fresh PBMCs (Exp. #1) or 14-day ZA/IL-2 stimulated Vγ9Vδ2 T cell cultures (Exp. #2-5) were grown without monocytes (effector, monocyte ratio 1:0) or 0.2-fold (4:1). ) or 5-fold (ratio 1:4) amount of monocytes ± 1 μM ZA. After 14 days the enrichment of Vγ9Vδ2 T cells in co-cultures was detected by multicolor FACS and proliferation of cultures was calculated. The enrichment factor of Vγ9Vδ2 T cells cultured at a ratio of 1:4 with monocytes was set to 100% in each experiment. The increase in monocytes in the cultures resulted in a more than 10-fold enrichment of Vγ9Vδ2 T cells. This effect was clearly ZA-dependent; cf. Figure 11a.

Additionally, human gastric cancer cells (NUGC-4-luciferase) and murine gastric cancer cells (CLS103 - calcein stained) were pretreated for 2 days in the presence or absence of 5 μM ZA. Human Vγ9Vδ2 T cells were MACS purified (day 14) and co-cultured with cancer cells for 24 hours. Cytotoxicity of Vγ9Vδ2 T cells against untreated and ZA-treated target cells was assessed by measuring residual luciferase activity or calcein fluorescence; cf. Figure 11b. target

Cells (NUGC-4 and CLS103) were pretreated for 2 days with or without 5 μM ZA followed by 4 h incubation with mitomycin c (50 MI) to stop proliferation. MACS purified human 14-day-old resting Vγ9Vδ2 T cells and ³ H-thymidine were added to the target cells and co-cultures were incubated at 37°C for 48 hours. Proliferation was detected by measuring ³ H thymidine incorporation in DNA using a MicroBeta scintillation counter. Proliferation of target cells w/o Vγ9Vδ2 T cells not treated with ZA was set at 100%; cf. Figure 11c.

As shown in Figures 11B and 11C, ZA-pulsed human cancer cells activated Vγ9Vδ2 T cells in terms of cytotoxicity (5-10-fold) and proliferation (1.4-1.8-fold), whereas the murine cancer cell line CLS103 activated Vγ9Vδ2 This effect was not elicited on T cells.

Example 9: ZA/IL-2 treatment affects the composition of PBMC cultures

Growth and differentiation of specific cell types in PBMC cultures depend on the presence of cytokines. These components are either added to the medium (e.g. growth factors present in serum, IL-2) or secreted by the immune cells themselves. Which type of cell develops also depends on the initial composition of the PBMC and genetic predisposition. To analyze the overall increase in effector cells (NK cells and Vγ9Vδ2 T cells), PBMCs from 10 different donors were grown for 14 days in the presence of 300 U/ml IL-2 and with or without 1 μM ZA. The amount of effector cells in the lymphocyte population was identified by multicolor FACS staining using CD3, CD16, CD56, Vγ9 and Vδ2 antibodies. CD3-CD56+CD16+ cells represent NK cells and CD3+Vγ9+Vδ2+ represent Vγ9Vδ2 T cells.

Multicolor FACS analysis revealed that NK cells mainly developed upon IL-2 treatment, whereas Vγ9Vδ2 T cells mainly proliferated in ZA/IL-2 treated cultures (FIG. 12).

Example 10: ZA/IL-2 treatment generates Vγ9Vδ2+ effector memory T cells

Subpopulations of T lymphocytes can be delineated using two surface markers: the chemokine receptor CCR7 and the high molecular weight isoform of the common lymphocyte antigen CD45RA. CCR7+ naive and centra-memory (CM) T cells are characterized by their ability to repeatedly circulate into lymph nodes and encounter antigens. In contrast, effector-memory (EM) and effector T lymphocytes RA+ (TEMRA) are characterized by downregulating CCR7 and migrating to peripheral nonlymphoid tissues, such as sites of infection or tumors. see. EM cells can also be further subdivided based on differential CD27 and CD28 expression. Progressive loss of CD28 and CD27 surface expression occurs concomitantly with upregulation of the cytolytic capacity of the cells. In addition, the level of CD57 correlates with the expression of granzymes and perforin and thus represents a third marker of cytotoxicity/cell maturation.

PBMCs were cultured in the presence or absence of 300 U/ml IL-2 and 1 μM ZA for 14 days. Expression of several different surface markers was detected by multicolor FACS analysis on days 0 and 14. Naive cells were CD45RA+ CCR7+, central memory cells (CM) were CD45RA- CCR7+, TEMRA were CD45RA+ CCR7-, and effector memory cells (EM) were negative for both markers; cf. Figure 13a. Additionally, cytolytic activity of Vγ9Vδ2 T-cells was detected by staining for CD27 and CD57 markers; cf. Figure 13b,c. In addition, the development of NK cell-like features important for ADCC activity was analyzed by staining CD3+ cells with CD16 (antibody binding) and CD56 (adherence); cf. Figure 13d.

Multicolor FACS analysis of Vγ9Vδ2 T cells showed that ZA/IL-2 treatment clearly promoted the development of CD27- and CD57+ EM-type Vγ9Vδ2 T cells (FIG. 13b-c). In addition to the enhanced cytolytic activity, increased levels of CD16 and CD56, known to be involved in ADCC from NK cells (CD3-CD16+CD56+), were observed in the CD3+ population (Figure 13D).

Taken together, these data suggest that ZA treatment of PBMCs results in the development of CD16+Vγ9+Vδ2+ effector memory T cells, which can migrate to peripheral non-lymphoid tissues and express markers of high cytolytic activity. In combination with the IMAB362 tumor targeting antibody, these cells can be extremely useful in mobilizing tumor cells to target and kill them.

Example 11: Z.A. /IL-2 extended Vγ9Vδ2 T cells IMAB362 - mediated CLDN18 .2 It is a powerful effector for dependency ADCC

Like NK cells, ZA/IL-2 expanded Vγ9Vδ2 T cells are positive for the FcγRIII receptor, CD16 (see Figures 9 and 13), via which cell-bound antibodies trigger ADCC. A series of experiments were conducted to evaluate whether Vγ9Vδ2 T cells could induce strong ADCC in association with IMAB362.

PBMCs derived from two different donors (#1 and #2) were cultured in medium supplemented with 300 U/ml IL-2 in the presence or absence of 1 μM ZA. After 14 days, cells were harvested and increasing concentrations of IMAB362 (0.26 ng/ml - 200 μg/ml) were added to NUGC-4 cells expressing CLDN18.2. Specific killing was detected in a luciferase assay; cf. Figure 14a. Figures 14B,C outline ADCC analysis performed on 27 donors grown at 300 U/ml IL-2 in the presence or absence of ZA. NUGC-4 functioned as a target cell. For each donor, the EC ₅₀ value calculated from the dose-response curve (b) and the maximum specific killing rate at 200 μg/ml IMAB362 (c) were scored in a scatter plot.

Strong IMAB362-dependent ADCC activity was observed for CLDN18.2-positive NUGC-4 cells using PBMCs cultured with ZA/IL-2 for 14 days (Figure 14a). Using ZA/IL-2-treated PBMC cultures, ADCC was dependent on the presence of Vγ9Vδ2 T cells (Figures 12 and 15). When cells are cultured in the absence of ZA, ADCC activity is reduced for most donors. In these cultures, residual ADCC activity was NK-cell dependent (Figures 11 and 14). After testing over 20 donors, ADCC analysis showed that ZA/IL-2 treatment of PBMCs improved EC ₅₀ and maximum specific killing compared to PBMCs cultured in the presence of IL-2 alone.

Additionally, PBMCs from two different donors (#1 + #2) were cultured with 1 μM ZA and 300 U/ml IL-2. This effector cell culture was used in ADCC analysis along with CLDN18.2-positive (NUGC-4, KATO III) and negative (SK-BR-3) human target cell lines (E:T ratio 40:1). Increasing amounts of IMAB362 antibody (0.26 ng/ml - 200 μg/ml) were added. ADCC was measured by luciferase assay; cf. Figure 15a. The same experiment as described in (a) was performed on effector cells and NUGC-4 target cells harvested from cultures treated with ZA/IL-2 at different time points; cf. Figure 15b. The same experiment as described in (a) was performed using NUGC-4 as target cells; cf. Figure 15c. ZA/IL-2 expanded cells were used directly, or Vγ9Vδ2 T cells were purified from cultures using TCRγδ MACS sorting (Miltenyi Biotech). Among lymphocytes, Vγ9Vδ2 T cells were obtained with a purity exceeding 97.0%.

Strong ADCC activity was observed for CLDN18.2-positive, but not CLDN18.2-negative human tumor cell lines (Figure 15A). Additionally, no ADCC activity was obtained with the isotype control antibody (not shown). During ZA/IL-2 treatment, ADCC lytic activity increases over time in a portion of the donors (Figure 15b). The dose/effect curve of IMAB362 shifts upward and to the left, indicating increased EC ₅₀ values and maximum dissolution rate over time. Compared to unconditioned PBMCs, Vγ9Vδ2 effector T cells enriched by ZA/IL-2 treatment can reach a higher maximum killing rate of CLDN18.2-positive target cells and in addition they have a lower killing rate for the same killing rate. Requires IMAB362

To confirm that Vγ9Vδ2 T cells are a reservoir for lytic activity, these cells were isolated from ZA/IL-2-cultured PBMC populations on day 14 at >97% purity by magnetic cell sorting. . In conjunction with IMAB362, ADCC activity was maintained and partially improved due to higher purity. These data confirm that Vγ9Vδ2 T cells are primarily responsible for the ADCC activity observed in 14-day-old PBMC media (Figure 15C).

Example 12: Z.A. /Treatment of target cell lines by IL-2 CLDN18 It does not affect the surface activity of .2.

The IMAB362 triggered mode of action is entirely dependent on the presence and amount of extracellular detectable CLDN18.2. Therefore, the effect of ZA/IL-2 treatment on CLDN18.2 surface density was analyzed by flow cytometry using NUGC-4 and KATO III cell lines expressing endogenous CLDN18.2. Specifically, flow cytometric analysis of IMAB362 binding to impermeable NUGC-4 gastric cancer cells pretreated with ZA/IL-2 or ZA/IL-2+EOF or ZA/IL-2+5-FU/OX for 72 h. carried out.

In vitro ZA/IL-2 treatment showed no change in the amount of CLDN18.2 surface localization; cf. Figure 16.

Example 13: of PBMCs Z.A. /by IL-2 treatment IMAB362 - mediated ADCC's increase is EOF Not weakened by pretreatment

Chemotherapeutic agents weaken cell proliferation. In contrast, ZA/IL-2 treatment triggers the expansion of Vγ9Vδ2 T cells. To analyze the impact of these opposing interactions on effector cells, PBMCs from six healthy donors were incubated with ZA/IL-2 or ZA/IL-2+EOF for 8 days prior to subjecting them to ADCC analysis. Cultured (E:T ratio 15:1). The IMAB362 concentration (EC ₅₀ ) that resulted in 50% ADCC-mediated lysis of untreated NUGC-4 target cells was determined.

The increase in IMAB362 induced ADCC of NUGC-4 cells in PBMCs resulting from treatment with ZA/IL-2 is not significantly altered by combined treatment of PBMCs with EOF (Figure 17).

Example 14: Nude in mouse human tumor cells In xenografts Anti-tumor effect of IMAB362 on and in vitro targeting of IMAB362 on CLDN18.2-positive tumors

80 μg Dyelight to investigate in vivo tumor cell targeting of IMAB362 The 80-labeled antibody was administered intravenously to nude mice subcutaneously xenografted with the human gastric cancer cell line NUGC-4. NUGC-4 cells showed surface expression of CLDN18.2 and HER2/neu (target of trastuzumab), but were negative for CD20. Dyelight in a group of NUGC-4 implanted mice 680-labeled trastuzumab (positive control) or Dyelight A control study was conducted by injecting either 680-labeled rituximab (negative control). Xenogen 24 hours after intravenous injection of antibody As demonstrated by real-time imaging of mice using a fluorescence imaging system, IMAB362 accumulated intensively and exclusively in mouse xenografts (Figure 18). IMAB362 remains effective in target-positive tumors even after 120 hours and is detectable with comparable intensity (Figure 18). Trastuzumab is also detected exclusively in xenografts 24 hours after injection. The trastuzumab signal rapidly disappears within 120 hours after injection. Rituximab signal is not detected.

In addition, nude mice producing CLDN18.2-positive xenograft tumors were treated using IMAB362. An initial treatment model study (IMAB362 administered 3 days after tumor cell inoculation) was conducted. Additionally, tumor treatment experiments were initiated when tumors reached a size of approximately 60-120 mm ³ , which lasted up to 9 days after tumor inoculation.

Nude mice were inoculated subcutaneously with 1x10 ⁷ HEK293~CLDN18.2 transfectants. Treatment was started for 10 mice per group 3 days after tumor inoculation. Mice were treated with 200 μg IMAB362, infliximab as an isotype control, and PBS twice a week for 6 weeks, alternating between intravenous and intraperitoneal administration routes. While all mice in the group treated with PBS or isotype control died within 70-80 days, animals treated with IMAB362 had a survival advantage (Figure 19). Not only was the time to death prolonged, but 4 out of 10 mice survived the entire 210-day observation period.

Treatment of 9 to 10 mice per group was initiated when the average tumor size reached 88 mm ³ (62 - 126 mm ³ ). Mice were measured in several test groups prior to treatment to ensure tumor size was comparable across all groups. Mice were treated alternately with 200 μg IMAB362, isotype control, or PBS by intravenous and intraperitoneal routes twice a week for 6 weeks. All mice in groups treated with PBS or isotype control died within 50-100 days. Animals treated with IMAB362 survived almost twice as long as the average survival time (47 days vs. 25 days). Three of these mice survived throughout the observation period (Figure 20). It is important to note that the in vivo antitumor effect depends on the presence of the target on tumor cells. No antitumor effect of IMAB362 treatment was observed in mice transplanted with CLDN18.2-negative HEK293 tumor cells.

The NUGC-4 gastric tumor model was used to investigate the efficacy of IMAB362 against cancer cells endogenously expressing CLDN18.2. NUGC-4 cells grow aggressively in nude mice.

1x10 ⁷ NUGC-4 gastric cancer cells were injected subcutaneously into the left flank of athymic nude mice (n = 9 for IMAB362 group; n = 8 for control group). Starting 6 days after intravenous injection of the tumor, IMAB362 (200 μg per injection) and control group were administered alternately intravenously and intraperitoneally twice a week. Tumor size was monitored twice a week. The data shown in Figure 21A are SEM averages. Tumor growth in mice treated with IMAB362 was significantly inhibited compared to mice treated with the control group (*p<0.05). Figure 21B shows tumor size at day 21 after tumor inoculation. The tumor size of IMAB362-treated mice was significantly smaller than that of control mice (*p<0.05).

When 1x10 ⁷ tumor cells are inoculated into mice, the median survival time of untreated mice is less than 25 days. Treatment with IMAB362, cetuximab, trastuzumab or isotype and buffer control was initiated when the average size of the tumor reached approximately 109 mm ³ (63 - 135 mm ³ ). Mice were stratified into treatment groups in a size-dependent manner (Figure 21). IMAB362 was shown to significantly reduce tumor growth rate. No significant reduction in tumor size was observed in this aggressively growing tumor model as compared to saline or antibody controls. Delay in tumor growth was associated with a non-significantly increased median survival time in IMAB362 treated mice (31 days vs. 25 days).

The antitumor activity of IMAB362 was evaluated in two human gastric cancer cases using NCI-N87 or NUGC-4 cells (NCI-N87~CLDN18.2 and NUGC-4~CLDN18.2) transduced lentivirally by IMAB362 target CLDN18.2. A paper xenograft model was tested.

NCI-N87~CLDN18.2 xenograft tumors were inoculated into the flanks of 8 nude mice (female, 6 weeks old) per treatment group by subcutaneous injection of 1x10 ⁷ NCI-N87~CLDN18.2 cells. Treatment was initiated 5 days after tumor inoculation by intravenously injecting 800 μg IMAB362 or 200 μl 0.9% NaCl for the saline control group. Intravenous administration was continued weekly during the entire observation period. Tumor size and animal health were measured semi-weekly. Figure 22A shows the effect of IMAB362 treatment on tumor growth. Subcutaneous tumor size was measured twice a week (mean + SEM, ***p<0.001). Figure 22b shows a Kaplan-Meier survival plot. Mice were sacrificed when tumor size reached 1400 mm ³ .

Accordingly, continuous IMAB362 treatment inhibited highly significant (p<0.001) tumor growth of NCI-N87~CLDN18.2 gastric carcinoma xenografts (FIG. 22A). Delay in tumor growth was associated with significantly (p<0.05) longer survival time in IMAB362 treated mice (Figure 22B).

The tumor size was significantly (p<0.05) smaller at 14 days after treatment of rapidly growing NUGC-4~CLDN18.2 xenografts with IMAB362 immunotherapy. After the first 2 weeks of IMAB362 treatment, tumor progression in NUGC-4~CLDN18.2 was very aggressive. However, by day 14 of treatment, NUGC-4~CLDN18.2 tumor growth was suppressed, significantly (p < 0.05) extending the survival period of IMAB362-treated mice.

In summary, IMAB362 was very effective in treating gastric carcinoma xenografts and was significant in slowing tumor progression and prolonging survival in an endogenous CLDN18.2-positive tumor model. Although this anti-tumor effect of IMAB362 in a highly aggressive tumor model system cannot be said to be very noticeable, it is nonetheless significant and demonstrates the strong anti-tumor efficacy of IMAB362.

Example 15: Combined with chemotherapy in mouse tumor model IMAB362's anti-tumor effect

In vitro , IMAB362-mediated ADCC is more effective in human gastric cancer cells pretreated with combinations of chemotherapy agents including EOF and 5-FU + OX. Therefore, the antitumor effect of IMAB362 in combination with these compounds was studied in vivo in a mouse tumor model.

NCI-N87~CLDN18.2 xenograft tumors were inoculated by subcutaneously injecting 1x10 ⁷ NCI-N87~CLDN18.2 cells into the flanks of 9 mice in each treatment group. Tumor-bearing mice were treated intraperitoneally with 1.25 mg/kg epirubicin, 3.25 mg/kg oxaliplatin, and 56.25 mg/kg 5-fluorouracil in combination with EOF therapy on days 4, 11, 18, and 25 after tumor inoculation. After treatment, 800 μg IMAB362 was injected intravenously 24 hours after chemotherapy administration. IMAB362 treatment was continued weekly. Tumor size and animal health were monitored twice a week. Figure 23A shows the effect of combination treatment on tumor growth. The size of subcutaneously administered tumors was measured twice a week (mean + SEM; * p < 0.05). Figure 23B shows Kaplan-Meier survival plot. Mice were sacrificed when tumor size reached 1400 mm ³ .

NCI-N87~CLDN18.2 tumor-producing nude mice treated with IMAB362 or EOF therapy showed highly significant inhibition of tumor growth compared to control mice. Additional IMAB362 treatment in combination with EOF chemotherapy resulted in significantly (p<0.05) higher tumor growth inhibition compared to treatment with EOF therapy alone (Figure 23A). The median survival time of mice in the saline control group was 59 days. In mice administered IMAB362 weekly, the median survival time was significantly extended to 76 days, which is similar to the median survival time of 76 days in the EOF group. However, when IMAB362 and EOF were combined, the median survival time increased to 81 days (FIG. 23b).

Ten nude mice (female, 6 weeks old) per treatment group were inoculated with xenograft tumors by subcutaneously injecting 1x10 ⁷ NUGC-4~CLDN18.2 cells into the flanks. Mice were treated with chemotherapy on days 3, 10, 17, and 24. IMAB362 treatment was continued weekly. Figure 24A shows tumor growth curves of subcutaneously administered NUGC-4~CLDN18.2 xenografts (mean + SEM). Figure 24B shows Kaplan-Meier survival plot (Log-rank (Mantel-Cox) Test, **p<0.01).

Subcutaneously administered NUGC-4~CLDN18.2 xenograft tumors grow very aggressively. Nonetheless, IMAB362 treatment of tumor-bearing nude mice significantly inhibited tumor growth compared to saline controls. In combination treatment with EOF, the effect of IMAB362 on NUGC-4~CLDN18.2 tumor growth was overshadowed by tumor inhibition due to EOF treatment, showing no significant increase in tumor growth inhibition compared to EOF treatment alone (FIG. 24a). However, the mean survival time of mice treated with IMAB362 and EOF therapy was significantly (p<0.01) prolonged compared to that of mice treated with EOF alone (FIG. 24B).

Example 16: Z.A. /IL-2 extended Vγ9Vδ2 T cells in vivo IMAB362-mediated regulation of advanced tumors. improve

To investigate the combined activity of IMAB362 and ZA/IL-2 producing γδ T cells in a mouse system, we used NSG mice. NSG mice lack mature T cells, B cells , natural killer (NK) cells, and multiple cytokine signaling pathways, so they have many deficits in innate immunity and niches in primary and secondary immunological tissues. This allows colonization by human immune cells.

1 x10 ⁷ to NSG mice CLDN18.2-transfected HEK293 cells were inoculated subcutaneously. On the same day, these mice were administered 8×10 ⁶ human PBMCs enriched for Vγ9Vδ2 T cells, cultured for 14 days in ZA-supplemented medium. Additionally, the mice were injected with 50 μg/kg ZA and 5000 U IL-2 (Proleukin). To maintain the function of human T cells, IL-2 was administered twice a week and ZA was administered weekly. When the HEK293-CLDN18.2 tumor grew large enough to be visible to the naked eye, weekly treatment with 200 μg IMAB362 was started. In addition to the 9 mice treated as described, 2 control groups of mice were established. One group was not administered human γδ T cells, and the other group was treated with an isotype control antibody instead of IMAB362. CLDN18.2-positive tumor outgrowth in mice treated with IMAB362 in the presence of human γδ T cells and ZA was significantly inhibited and almost abolished, whereas mice treated with isotype control antibodies or lacking human T cell effectors In these cases, the tumors grew aggressively and the mice had to be sacrificed early (FIG. 25).

Example 17: Combined with chemotherapy in mouse tumor model IMAB362's anti-tumor effect

In subcutaneous gastric carcinoma allografts in immunocompetitive outbred NMRI mice (CLS-103~cldn18.2) using CLS-103 cells with lentiviral transduction of murine cldn18.2, combined with chemotherapy. The antitumor activity of IMAB362 was tested.

CLS-103~cldn18.2 allograft tumors were inoculated by subcutaneously injecting 1x10 ⁶ CLS-103~cldn18.2 cells into the flanks of 10 NMRI mice per treatment group. Tumor-bearing mice were treated with 1.25 mg/kg epirubicin, 3.25 mg/kg oxaliplatin, and 56.25 mg/kg 5-fluorouracil (EOF) on days 3, 10, 17, and 24 after tumor inoculation, followed by treatment with each chemical. 24 hours after administration of the therapy, 800 μg IMAB362 was injected intravenously. IL-2 was administered by subcutaneous injection of 3000 IE twice a week. After completion of chemotherapy, IMAB362 and IL-2 treatment continued throughout the observation period. Tumor size and animal health were monitored twice a week. Mice were sacrificed when tumor size reached 1400 mm ³ or when tumors became ulcerous.

As shown in Figure 26, CLS-103~cldn18.2 tumor-producing NMRI mice treated with IMAB362 or EOF alone did not show significant tumor growth inhibition compared to the saline control group. In contrast, the combination of EOF chemotherapy and IMAB362 treatment resulted in significantly greater inhibition of tumor growth and significantly prolonged survival of tumor-bearing mice. These observations indicate that the combination of EOF chemotherapy and IMAB362 immunotherapy produces additive or even synergistic therapeutic effects. IL-2 treatment had no effect on tumor growth.

New international patent application

Ganymed Pharmaceuticals AG, et al.

“Combination therapy associated with antibodies to claudin 18.2 for cancer treatment”

Party Clearance number: 342-71 PCT

Supplementary Sheet for Biological Agents

Additional deposit indications:

1) The names and addresses of the depositories of the depositing agents (DSM ACC2738, DSM ACC2739, DSM ACC2740, DSM ACC2741, DSM ACC2742, DSM ACC2743, DSM ACC-2745, DSM ACC2746, DSM ACC2747, DSM ACC2748) are as follows:

DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH

Macheroder Weg 1B, Braunschweig, Germany 38124

2) The names and addresses of the depositories for the depository agents (DSM ACC2808, DSM ACC2809, DSM ACC2810) are as follows:

DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH

Germany 38124 Braunschweig Inhofenstrasse 7be

Additional comments applicable to all of the above deposited microorganisms:

- Mouse (Mus musculus) myeloma fused with mouse (Mus musculus) spleen cells P3X63Ag8U.1

- Hybridoma secreting antibodies to human claudin-18A2

3) Depositor:

All of the foregoing deposits were made by the following depositors:

Ganymed Pharmaceuticals AG

Freiligratstrasse 12, Mainz, 55131 Germany

DE

Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH DSMACC2737 20051019 Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH DSMACC2738 20051019 Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH DSMACC2739 20051019 Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH DSMACC2740 20051019 Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH DSMACC2741 20051019 Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH DSMACC2742 20051019 Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH DSMACC2743 20051019 Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH DSMACC2745 20051117 Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH DSMACC2746 20051117 Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH DSMACC2747 20051117 Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH DSMACC2748 20051117 Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH DSMACC2808 20061026 Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH DSMACC2809 20061026 Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH DSMACC2810 20061026

SEQUENCE LISTING <110> Ganymed Pharmaceuticals AG <120> COMBINATION THERAPY INVOLVING ANTIBODIES AGAINST CLAUDIN 18.2 FOR TREATMENT OF CANCER <130> 342-71 PCT <150> PCT/EP2012/002210 <151> 2012-05-23 <160> 50 < 170> PatentIn version 3.5 <210> 1 <211> 261 <212> PRT <213> Homo sapiens <400> 1 Met Ala Val Thr Ala Cys Gln Gly Leu Gly Phe Val Val Ser Leu Ile 1 5 10 15 Gly Ile Ala Gly Ile Ile Ala Ala Thr Cys Met Asp Gln Trp Ser Thr 20 25 30 Gln Asp Leu Tyr Asn Asn Pro Val Thr Ala Val Phe Asn Tyr Gln Gly 35 40 45 Leu Trp Arg Ser Cys Val Arg Glu Ser Ser Gly Phe Thr Glu Cys Arg 50 55 60 Gly Tyr Phe Thr Leu Leu Gly Leu Pro Ala Met Leu Gln Ala Val Arg 65 70 75 80 Ala Leu Met Ile Val Gly Ile Val Leu Gly Ala Ile Gly Leu Leu Val 85 90 95 Ser Ile Phe Ala Leu Lys Cys Ile Arg Ile Gly Ser Met Glu Asp Ser 100 105 110 Ala Lys Ala Asn Met Thr Leu Thr Ser Gly Ile Met Phe Ile Val Ser 115 120 125 Gly Leu Cys Ala Ile Ala Gly Val Ser Val Phe Ala Asn Met Leu Val 130 135 140 Thr Asn Phe Trp Met Ser Thr Ala Asn Met Tyr Thr Gly Met Gly Gly 145 150 155 160 Met Val Gln Thr Val Gln Thr Arg Tyr Thr Phe Gly Ala Ala Leu Phe 165 170 175 Val Gly Trp Val Ala Gly Gly Leu Thr Leu Ile Gly Gly Val Met Met 180 185 190 Cys Ile Ala Cys Arg Gly Leu Ala Pro Glu Glu Thr Asn Tyr Lys Ala 195 200 205 Val Ser Tyr His Ala Ser Gly His Ser Val Ala Tyr Lys Pro Gly Gly 210 215 220 Phe Lys Ala Ser Thr Gly Phe Gly Ser Asn Thr Lys Asn Lys Lys Ile 225 230 235 240 Tyr Asp Gly Gly Ala Arg Thr Glu Asp Glu Val Gln Ser Tyr Pro Ser 245 250 255 Lys His Asp Tyr Val 260 <210> 2 <211> 261 <212 > PRT <213> Homo sapiens <400> 2 Met Ser Thr Thr Thr Cys Gln Val Val Ala Phe Leu Leu Ser Ile Leu 1 5 10 15 Gly Leu Ala Gly Cys Ile Ala Ala Thr Gly Met Asp Met Trp Ser Thr 20 25 30 Gln Asp Leu Tyr Asp Asn Pro Val Thr Ser Val Phe Gln Tyr Glu Gly 35 40 45 Leu Trp Arg Ser Cys Val Arg Gln Ser Ser Gly Phe Thr Glu Cys Arg 50 55 60 Pro Tyr Phe Thr Ile Leu Gly Leu Pro Ala Met Leu Gln Ala Val Arg 65 70 75 80 Ala Leu Met Ile Val Gly Ile Val Leu Gly Ala Ile Gly Leu Leu Val 85 90 95 Ser Ile Phe Ala Leu Lys Cys Ile Arg Ile Gly Ser Met Glu Asp Ser 100 105 110 Ala Lys Ala Asn Met Thr Leu Thr Ser Gly Ile Met Phe Ile Val Ser 115 120 125 Gly Leu Cys Ala Ile Ala Gly Val Ser Val Phe Ala Asn Met Leu Val 130 135 140 Thr Asn Phe Trp Met Ser Thr Ala Asn Met Tyr Thr Gly Met Gly Gly 145 150 155 160 Met Val Gln Thr Val Gln Thr Arg Tyr Thr Phe Gly Ala Ala Leu Phe 165 170 175 Val Gly Trp Val Ala Gly Gly Leu Thr Leu Ile Gly Gly Val Met Met 180 185 190 Cys Ile Ala Cys Arg Gly Leu Ala Pro Glu Glu Thr Asn Tyr Lys Ala 195 200 205 Val Ser Tyr His Ala Ser Gly His Ser Val Ala Tyr Lys Pro Gly Gly 210 215 220 Phe Lys Ala Ser Thr Gly Phe Gly Ser Asn Thr Lys Asn Lys Lys Ile 225 230 235 240 Tyr Asp Gly Gly Ala Arg Thr Glu Asp Glu Val Gln Ser Tyr Pro Ser 245 250 255 Lys His Asp Tyr Val 260 <210> 3 <211> 10 <212> PRT <213> Homo sapiens <400> 3 Asp Gln Trp Ser Thr Gln Asp Leu Tyr Asn 1 5 10 <210> 4 <211> 11 <212> PRT <213> Homo sapiens <400> 4 Asn Asn Pro Val Thr Ala Val Phe Asn Tyr Gln 1 5 10 <210> 5 <211 > 14 <212> PRT <213> Homo sapiens <400> 5 Ser Thr Gln Asp Leu Tyr Asn Asn Pro Val Thr Ala Val Phe 1 5 10 <210> 6 <211> 13 <212> PRT <213> Homo sapiens < 400> 6 Thr Asn Phe Trp Met Ser Thr Ala Asn Met Tyr Thr Gly 1 5 10 <210> 7 <211> 13 <212> PRT <213> Homo sapiens <400> 7 Asp Ser Ala Lys Ala Asn Met Thr Leu Thr Ser Gly Ile 1 5 10 <210> 8 <211> 55 <212> PRT <213> Homo sapiens <400> 8 Met Asp Gln Trp Ser Thr Gln Asp Leu Tyr Asn Asn Pro Val Thr Ala 1 5 10 15 Val Phe Asn Tyr Gln Gly Leu Trp Arg Ser Cys Val Arg Glu Ser Ser 20 25 30 Gly Phe Thr Glu Cys Arg Gly Tyr Phe Thr Leu Leu Gly Leu Pro Ala 35 40 45 Met Leu Gln Ala Val Arg Ala 50 55 <210> 9 <211 > 24 <212> PRT <213> Homo sapiens <400> 9 Phe Ala Leu Lys Cys Ile Arg Ile Gly Ser Met Glu Asp Ser Ala Lys 1 5 10 15 Ala Asn Met Thr Leu Thr Ser Gly 20 <210> 10 <211 > 40 <212> PRT <213> Homo sapiens <400> 10 Ala Asn Met Leu Val Thr Asn Phe Trp Met Ser Thr Ala Asn Met Tyr 1 5 10 15 Thr Gly Met Gly Gly Met Val Gln Thr Val Gln Thr Arg Tyr Thr Phe 20 25 30 Gly Ala Ala Leu Phe Val Gly Trp 35 40 <210> 11 <211> 153 <212> PRT <213> Homo sapiens <400> 11 Met Asp Gln Trp Ser Thr Gln Asp Leu Tyr Asn Asn Pro Val Thr Ala 1 5 10 15 Val Phe Asn Tyr Gln Gly Leu Trp Arg Ser Cys Val Arg Glu Ser Ser 20 25 30 Gly Phe Thr Glu Cys Arg Gly Tyr Phe Thr Leu Leu Gly Leu Pro Ala 35 40 45 Met Leu Gln Ala Val Arg Ala Leu Met Ile Val Gly Ile Val Leu Gly 50 55 60 Ala Ile Gly Leu Leu Val Ser Ile Phe Ala Leu Lys Cys Ile Arg Ile 65 70 75 80 Gly Ser Met Glu Asp Ser Ala Lys Ala Asn Met Thr Leu Thr Ser Gly 85 90 95 Ile Met Phe Ile Val Ser Gly Leu Cys Ala Ile Ala Gly Val Ser Val 100 105 110 Phe Ala Asn Met Leu Val Thr Asn Phe Trp Met Ser Thr Ala Asn Met 115 120 125 Tyr Thr Gly Met Gly Gly Met Val Gln Thr Val Gln Thr Arg Tyr Thr 130 135 140 Phe Gly Ala Ala Leu Phe Val Gly Trp 145 150 <210> 12 <211> 107 <212> PRT <213> Artificial <220> <223> Description of artificial sequence: Translation of PCR product <400> 12 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 13 <211> 326 <212> PRT <213> Artificial < 220> <223> Description of artificial sequence: Translation of PCR product <400> 13 Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly 1 5 10 15 Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 20 25 30 Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 35 40 45 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 50 55 60 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn 65 70 75 80 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro 85 90 95 Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 100 105 110 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 115 120 125 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 130 135 140 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 145 150 155 160 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 165 170 175 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 180 185 190 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 195 200 205 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 210 215 220 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn 225 230 235 240 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 245 250 255 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 260 265 270 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 275 280 285 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 290 295 300 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 305 310 315 320 Ser Leu Ser Pro Gly Lys 325 <210> 14 <211> 466 <212> PRT <213> Artificial <220> <223> Description of artificial sequence: chimeric monoclonal antibody <400> 14 Met Glu Trp Thr Trp Val Phe Leu Phe Leu Leu Ser Val Thr Ala Gly 1 5 10 15 Val His Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Met Lys 20 25 30 Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Thr Gly Tyr Thr Phe 35 40 45 Ser Ser Tyr Trp Ile Glu Trp Val Lys Gln Arg Pro Gly His Gly Leu 50 55 60 Glu Trp Ile Gly Glu Ile Leu Pro Gly Ser Gly Ser Thr Asn Tyr Asn 65 70 75 80 Glu Lys Phe Lys Gly Lys Ala Thr Phe Thr Ala Asp Thr Ser Ser Asn 85 90 95 Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Tyr Asp Tyr Pro Trp Phe Ala Tyr Trp Gly Gln 115 120 125 Gly Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val 130 135 140 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 145 150 155 160 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 165 170 175 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 180 185 190 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 195 200 205 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 210 215 220 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 225 230 235 240 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 245 250 255 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 260 265 270 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 275 280 285 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 290 295 300 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 305 310 315 320 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 325 330 335 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 340 345 350 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 355 360 365 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 370 375 380 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 385 390 395 400 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Pro Val 405 410 415 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 420 425 430 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 435 440 445 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 450 455 460 Gly Lys 465 <210> 15 <211> 467 <212> PRT <213> Artificial <220> <223> Description of artificial sequence: chimeric monoclonal antibody <400> 15 Met Asp Trp Leu Trp Asn Leu Leu Phe Leu Met Ala Ala Ala Gln Ser 1 5 10 15 Ile Gln Ala Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys 20 25 30 Pro Gly Glu Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 Thr Asn Tyr Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu 50 55 60 Lys Trp Met Gly Trp Ile Asn Thr Asn Thr Gly Glu Pro Thr Tyr Ala 65 70 75 80 Glu Glu Phe Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser 85 90 95 Thr Ala Tyr Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr 100 105 110 Tyr Phe Cys Ala Arg Leu Gly Phe Gly Asn Ala Met Asp Tyr Trp Gly 115 120 125 Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 130 135 140 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 145 150 155 160 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 165 170 175 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 180 185 190 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 195 200 205 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 210 215 220 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 225 230 235 240 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 245 250 255 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 260 265 270 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 275 280 285 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 290 295 300 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 305 310 315 320 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 325 330 335 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 340 345 350 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 355 360 365 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 370 375 380 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 385 390 395 400 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 405 410 415 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 420 425 430 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 435 440 445 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 450 455 460 Pro Gly Lys 465 < 210> 16 <211> 465 <212> PRT <213> Artificial <220> <223> Description of artificial sequence: chimeric monoclonal antibody <400> 16 Met Glu Trp Ile Trp Ile Phe Leu Phe Ile Leu Ser Gly Thr Ala Gly 1 5 10 15 Val His Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg 20 25 30 Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 Thr Asp Tyr Tyr Ile Asn Trp Val Lys Gln Arg Thr Gly Gln Gly Leu 50 55 60 Glu Trp Ile Gly Glu Ile Tyr Pro Gly Ser Gly Asn Thr Tyr Tyr Asn 65 70 75 80 Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser 85 90 95 Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val 100 105 110 Tyr Phe Cys Ala Arg Ser Tyr Gly Ala Phe Asp Tyr Trp Gly Gln Gly 115 120 125 Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 130 135 140 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 145 150 155 160 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 165 170 175 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 180 185 190 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 195 200 205 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 210 215 220 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 225 230 235 240 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 245 250 255 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 260 265 270 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 275 280 285 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 290 295 300 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 305 310 315 320 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 325 330 335 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 340 345 350 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 355 360 365 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 370 375 380 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 385 390 395 400 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Pro Val Leu 405 410 415 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 420 425 430 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 435 440 445 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 450 455 460 Lys 465 <210> 17 <211> 467 <212> PRT <213> Artificial <220> <223> Description of artificial sequence: chimeric monoclonal antibody <400> 17 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg 20 25 30 Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 Thr Ser Tyr Trp Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu 50 55 60 Glu Trp Ile Gly Asn Ile Tyr Pro Ser Asp Ser Tyr Thr Asn Tyr Asn 65 70 75 80 Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser 85 90 95 Thr Ala Tyr Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val 100 105 110 Tyr Tyr Cys Thr Arg Ser Trp Arg Gly Asn Ser Phe Asp Tyr Trp Gly 115 120 125 Gln Gly Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 130 135 140 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 145 150 155 160 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 165 170 175 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 180 185 190 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 195 200 205 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 210 215 220 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 225 230 235 240 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 245 250 255 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 260 265 270 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 275 280 285 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 290 295 300 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 305 310 315 320 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 325 330 335 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 340 345 350 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 355 360 365 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 370 375 380 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 385 390 395 400 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Pro 405 410 415 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 420 425 430 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 435 440 445 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 450 455 460 Pro Gly Lys 465 <210> 18 <211> 466 <212> PRT <213> Artificial <220> <223> Description of artificial sequence: chimeric monoclonal antibody <400> 18 Met Glu Trp Arg Ile Phe Leu Phe Ile Leu Ser Gly Thr Ala Gly Val 1 5 10 15 His Ser Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro 20 25 30 Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr 35 40 45 Asp Tyr Val Ile Ser Trp Val Lys Gln Arg Thr Gly Gln Gly Leu Glu 50 55 60 Trp Ile Gly Glu Ile Tyr Pro Gly Ser Gly Ser Thr Tyr Tyr Asn Glu 65 70 75 80 Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Asn Thr 85 90 95 Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr 100 105 110 Phe Cys Ala Arg Gly Val Leu Leu Arg Ala Met Asp Tyr Trp Gly Gln 115 120 125 Gly Thr Ser Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 130 135 140 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 145 150 155 160 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 165 170 175 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 180 185 190 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 195 200 205 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 210 215 220 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 225 230 235 240 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 245 250 255 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 260 265 270 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 275 280 285 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 290 295 300 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 305 310 315 320 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 325 330 335 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 340 345 350 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 355 360 365 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 370 375 380 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 385 390 395 400 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 405 410 415 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 420 425 430 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 435 440 445 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 450 455 460 Gly Lys 465 <210> 19 <211> 469 <212> PRT <213> Artificial <220> <223> Description of artificial sequence: chimeric monoclonal antibody <400> 19 Met Asp Trp Ile Trp Ile Met Leu His Leu Leu Ala Ala Ala Thr Gly 1 5 10 15 Ile Gln Ser Gln Val His Leu Gln Gln Ser Gly Ser Glu Leu Arg Ser 20 25 30 Pro Gly Ser Ser Val Lys Leu Ser Cys Lys Asp Phe Asp Ser Glu Val 35 40 45 Phe Pro Phe Ala Tyr Met Ser Trp Ile Arg Gln Lys Pro Gly His Gly 50 55 60 Phe Glu Trp Ile Gly Asp Ile Leu Pro Ser Ile Gly Arg Thr Ile Tyr 65 70 75 80 Gly Glu Lys Phe Glu Asp Lys Ala Thr Leu Asp Ala Asp Thr Val Ser 85 90 95 Asn Thr Ala Tyr Leu Glu Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala 100 105 110 Ile Tyr Tyr Cys Ala Arg Gly Glu Gly Tyr Gly Ala Trp Phe Ala Tyr 115 120 125 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly 130 135 140 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 145 150 155 160 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 165 170 175 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 180 185 190 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 195 200 205 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 210 215 220 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 225 230 235 240 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 245 250 255 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 260 265 270 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 275 280 285 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 290 295 300 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 305 310 315 320 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 325 330 335 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 340 345 350 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 355 360 365 Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln 370 375 380 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 385 390 395 400 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 405 410 415 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 420 425 430 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 435 440 445 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 450 455 460 Leu Ser Pro Gly Lys 465 <210> 20 <211> 240 <212> PRT <213> Artificial <220> <223> Description of artificial sequence: chimeric monoclonal antibody <400> 20 Met Glu Ser Gln Thr Gln Val Leu Met Ser Leu Leu Phe Trp Val Ser 1 5 10 15 Gly Thr Cys Gly Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr 20 25 30 Val Thr Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser 35 40 45 Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln 50 55 60 Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg 65 70 75 80 Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp 85 90 95 Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr 100 105 110 Tyr Cys Gln Asn Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr 115 120 125 Lys Leu Glu Leu Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe 130 135 140 Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys 145 150 155 160 Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val 165 170 175 Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln 180 185 190 Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser 195 200 205 Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His 210 215 220 Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 240 <210> 21 <211> 235 <212> PRT < 213> Artificial <220> <223> Description of artificial sequence: chimeric monoclonal antibody <400> 21 Met His Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser 1 5 10 15 Val Ile Met Ser Arg Gly Gln Ile Val Leu Thr Gln Ser Pro Ala Ile 20 25 30 Met Ser Ala Ser Pro Gly Glu Lys Val Thr Ile Thr Cys Ser Ala Ser 35 40 45 Ser Ser Val Ser Tyr Met His Trp Phe Gln Gln Lys Pro Gly Thr Ser 50 55 60 Pro Lys Leu Trp Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro 65 70 75 80 Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile 85 90 95 Ser Arg Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Arg 100 105 110 Ser Ser Tyr Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 115 120 125 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 130 135 140 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 145 150 155 160 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 165 170 175 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 180 185 190 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 195 200 205 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 210 215 220 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 <210> 22 <211> 234 <212> PRT <213> Artificial <220> <223> Description of artificial sequence: chimeric monoclonal antibody <400> 22 Met Glu Phe Gln Thr Gln Val Phe Val Phe Val Leu Leu Trp Leu Ser 1 5 10 15 Gly Val Asp Gly Asp Ile Val Met Thr Gln Ser Gln Lys Phe Met Ser 20 25 30 Thr Ser Val Gly Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asn 35 40 45 Val Arg Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro 50 55 60 Lys Ala Leu Ile Tyr Leu Ala Ser Asn Arg His Thr Gly Val Pro Asp 65 70 75 80 Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 85 90 95 Asn Val Gln Ser Glu Asp Leu Ala Asp Tyr Phe Cys Leu Gln His Trp 100 105 110 Asn Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 115 120 125 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 130 135 140 Leu Lys Ser Gly Thr Ala Ser Val Val Val Cys Leu Leu Asn Asn Phe Tyr 145 150 155 160 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 165 170 175 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 180 185 190 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 195 200 205 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 210 215 220 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 <210> 23 <211> 240 <212> PRT <213> Artificial <220> <223> Description of artificial sequence: chimeric monoclonal antibody < 400>23 Met Asp Ser Gln Ala Gln Val Leu Met Leu Leu Leu Leu Trp Val Ser 1 5 10 15 Gly Thr Cys Gly Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala 20 25 30 Val Ser Val Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser 35 40 45 Leu Leu Tyr Ser Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln 50 55 60 Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg 65 70 75 80 Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp 85 90 95 Phe Thr Leu Thr Ile Ser Ser Val Lys Ala Glu Asp Leu Ala Val Tyr 100 105 110 Tyr Cys Gln Gln Tyr Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr 115 120 125 Lys Leu Glu Leu Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe 130 135 140 Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys 145 150 155 160 Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val 165 170 175 Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln 180 185 190 Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser 195 200 205 Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His 210 215 220 Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 240 <210> 24 <211> 240 <212> PRT <213> Artificial <220> <223> Description of artificial sequence: chimeric monoclonal antibody <400> 24 Met Glu Ser Gln Thr Gln Val Leu Met Ser Leu Leu Phe Trp Val Ser 1 5 10 15 Gly Thr Cys Gly Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr 20 25 30 Val Thr Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser 35 40 45 Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln 50 55 60 Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg 65 70 75 80 Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp 85 90 95 Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr 100 105 110 Tyr Cys Gln Asn Asp Tyr Ser Tyr Pro Phe Thr Phe Gly Ser Gly Thr 115 120 125 Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe 130 135 140 Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys 145 150 155 160 Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val 165 170 175 Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln 180 185 190 Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser 195 200 205 Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His 210 215 220 Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 240 <210> 25 <211> 239 <212> PRT <213> Artificial <220> <223> Description of artificial sequence: chimeric monoclonal antibody <400> 25 Met Asp Ser Gln Ala Gln Val Leu Ile Leu Leu Leu Leu Trp Val Ser 1 5 10 15 Gly Thr Cys Gly Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala 20 25 30 Val Ser Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser 35 40 45 Leu Leu Asn Ser Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln 50 55 60 Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg 65 70 75 80 Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp 85 90 95 Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr 100 105 110 Tyr Cys Lys Gln Ser Tyr Asn Leu Tyr Thr Phe Gly Gly Gly Thr Lys 115 120 125 Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro 130 135 140 Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu 145 150 155 160 Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp 165 170 175 Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp 180 185 190 Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys 195 200 205 Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln 210 215 220 Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 <210> 26 <211> 240 <212> PRT <213> Artificial < 220> <223> Description of artificial sequence: chimeric monoclonal antibody <400> 26 Met Asp Ser Gln Ala Gln Val Leu Met Leu Leu Leu Leu Trp Val Ser 1 5 10 15 Gly Thr Cys Gly Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala 20 25 30 Val Ser Val Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser 35 40 45 Leu Leu Tyr Ser Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln 50 55 60 Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg 65 70 75 80 Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Ala Thr Asp 85 90 95 Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Asp Tyr 100 105 110 His Cys Gly Gln Gly Tyr Ser Tyr Pro Tyr Thr Phe Gly Gly Gly Thr 115 120 125 Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe 130 135 140 Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys 145 150 155 160 Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val 165 170 175 Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln 180 185 190 Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser 195 200 205 Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His 210 215 220 Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 240 <210> 27 <211> 240 <212> PRT <213> Artificial <220> <223> Description of artificial sequence: chimeric monoclonal antibody <400> 27 Met Asp Ser Gln Ala Gln Val Leu Met Leu Leu Leu Leu Trp Val Ser 1 5 10 15 Gly Thr Cys Gly Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala 20 25 30 Val Ser Val Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser 35 40 45 Leu Leu Tyr Ser Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Gln 50 55 60 Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg 65 70 75 80 Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp 85 90 95 Phe Thr Leu Thr Ile Ser Ser Val Lys Ala Glu Asp Leu Ala Val Tyr 100 105 110 Tyr Cys Gln Gln Tyr Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr 115 120 125 Lys Leu Glu Leu Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe 130 135 140 Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys 145 150 155 160 Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val 165 170 175 Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln 180 185 190 Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser 195 200 205 Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His 210 215 220 Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 240 <210> 28 <211> 234 <212> PRT <213> Artificial <220> <223 > Description of artificial sequence: chimeric monoclonal antibody <400> 28 Met Glu Ser Gln Thr Leu Val Phe Ile Ser Ile Leu Leu Trp Leu Tyr 1 5 10 15 Gly Ala Asp Gly Asn Ile Val Met Thr Gln Ser Pro Lys Ser Met Ser 20 25 30 Met Ser Val Gly Glu Arg Val Thr Leu Thr Cys Lys Ala Ser Glu Asn 35 40 45 Val Val Thr Tyr Val Ser Trp Tyr Gln Gln Lys Pro Glu Gln Ser Pro 50 55 60 Lys Leu Leu Ile Tyr Gly Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp 65 70 75 80 Arg Phe Thr Gly Ser Gly Ser Ala Thr Asp Phe Thr Leu Thr Ile Ser 85 90 95 Ser Val Lys Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln Tyr Tyr 100 105 110 Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg 115 120 125 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Ser Asp Glu Gln 130 135 140 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 145 150 155 160 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 165 170 175 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 180 185 190 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 195 200 205 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 210 215 220 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 <210> 29 <211 > 117 <212> PRT <213> Artificial <220> <223> Description of artificial sequence: Translation of PCR product <400> 29 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Met Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Thr Gly Tyr Thr Phe Ser Ser Tyr 20 25 30 Trp Ile Glu Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Leu Pro Gly Ser Gly Ser Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Phe Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Asp Tyr Pro Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ala 115 <210> 30 <211> 118 <212> PRT <213> Artificial <220> <223> Description of artificial sequence: Translation of PCR product <400> 30 Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met 35 40 45 Gly Trp Ile Asn Thr Asn Thr Gly Glu Pro Thr Tyr Ala Glu Glu Phe 50 55 60 Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95 Ala Arg Leu Gly Phe Gly Asn Ala Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Ser Val Thr Val Ser Ser 115 <210> 31 <211> 116 <212> PRT <213> Artificial <220> <223> Description of artificial sequence: Translation of PCR product <400> 31 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Tyr Ile Asn Trp Val Lys Gln Arg Thr Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Tyr Pro Gly Ser Gly Asn Thr Tyr Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Ser Tyr Gly Ala Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu 100 105 110 Thr Val Ser Ser 115 <210> 32 <211> 118 <212> PRT <213> Artificial <220> <223> Description of artificial sequence: Translation of PCR product <400> 32 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asn Ile Tyr Pro Ser Asp Ser Tyr Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Ser Trp Arg Gly Asn Ser Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Leu Thr Val Ser Ser 115 < 210> 33 <211> 118 <212> PRT <213> Artificial <220> <223> Description of artificial sequence: Translation of PCR product <400> 33 Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Val Ile Ser Trp Val Lys Gln Arg Thr Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Tyr Pro Gly Ser Gly Ser Thr Tyr Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Asn Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Gly Val Leu Leu Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Ser Val Thr Val Ser Ser 115 <210> 34 <211> 120 <212> PRT <213> Artificial <220> <223> Description of artificial sequence: Translation of PCR product <400> 34 Gln Val His Leu Gln Gln Ser Gly Ser Glu Leu Arg Ser Pro Gly Ser 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Asp Phe Asp Ser Glu Val Phe Pro Phe 20 25 30 Ala Tyr Met Ser Trp Ile Arg Gln Lys Pro Gly His Gly Phe Glu Trp 35 40 45 Ile Gly Asp Ile Leu Pro Ser Ile Gly Arg Thr Ile Tyr Gly Glu Lys 50 55 60 Phe Glu Asp Lys Ala Thr Leu Asp Ala Asp Thr Val Ser Asn Thr Ala 65 70 75 80 Tyr Leu Glu Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Ile Tyr Tyr 85 90 95 Cys Ala Arg Gly Glu Gly Tyr Gly Ala Trp Phe Ala Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ala 115 120 <210> 35 <211> 113 <212> PRT <213> Artificial <220> <223> Description of artificial sequence: Translation of PCR product <400> 35 Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu 100 105 110 Lys <210> 36 <211> 106 <212> PRT <213> Artificial <220> <223> Description of artificial sequence: Translation of PCR product <400> 36 Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 His Trp Phe Gln Gln Lys Pro Gly Thr Ser Pro Lys Leu Trp Ile Tyr 35 40 45 Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Met Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Tyr Pro Pro Thr 85 90 95 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 37 <211> 107 <212 > PRT <213> Artificial <220> <223> Description of artificial sequence: Translation of PCR product <400> 37 Asp Ile Val Met Thr Gln Ser Gln Lys Phe Met Ser Thr Ser Val Gly 1 5 10 15 Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asn Val Arg Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Ala Leu Ile 35 40 45 Tyr Leu Ala Ser Asn Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser 65 70 75 80 Glu Asp Leu Ala Asp Tyr Phe Cys Leu Gln His Trp Asn Tyr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 38 <211> 113 <212> PRT <213> Artificial <220> <223> Description of artificial sequence: Translation of PCR product <400> 38 Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Val Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30 Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Lys Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln 85 90 95 Tyr Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu 100 105 110 Lys <210> 39 <211> 113 <212> PRT <213> Artificial <220> <223> Description of artificial sequence: Translation of PCR product <400> 39 Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asp Tyr Ser Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile 100 105 110 Lys <210> 40 <211> 112 <212> PRT <213> Artificial <220> <223> Description of artificial sequence: Translation of PCR product <400> 40 Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Ala Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Lys Gln 85 90 95 Ser Tyr Asn Leu Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 41 <211> 113 <212> PRT <213> Artificial <220> < 223> Description of artificial sequence: Translation of PCR product <400> 41 Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Val Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30 Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Ala Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Asp Tyr His Cys Gly Gln 85 90 95 Gly Tyr Ser Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 100 105 110 Lys <210> 42 <211> 113 <212> PRT <213> Artificial <220> <223> Description of artificial sequence: Translation of PCR product <400> 42 Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Val Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30 Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Lys Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln 85 90 95 Tyr Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu 100 105 110 Lys <210> 43 <211> 107 <212> PRT <213> Artificial <220> <223> Description of artificial sequence: Translation of PCR product <400> 43 Asn Ile Val Met Thr Gln Ser Pro Lys Ser Met Ser Met Ser Val Gly 1 5 10 15 Glu Arg Val Thr Leu Thr Cys Lys Ala Ser Glu Asn Val Val Thr Tyr 20 25 30 Val Ser Trp Tyr Gln Gln Lys Pro Glu Gln Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Ser Ala Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Lys Ala 65 70 75 80 Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln Tyr Tyr Ser Tyr Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 <210> 44 <211> 15 <212> PRT <213 > Artificial <220> <223> Epitope <400> 44 Met Asp Gln Trp Ser Thr Gln Asp Leu Tyr Asn Asn Pro Val Thr 1 5 10 15 <210> 45 <211> 15 <212> PRT <213> Artificial <220 > <223> Epitope <400> 45 Ser Thr Gln Asp Leu Tyr Asn Asn Pro Val Thr Ala Val Phe Asn 1 5 10 15 <210> 46 <211> 15 <212> PRT <213> Artificial <220> <223> Epitope <400> 46 Leu Tyr Asn Asn Pro Val Thr Ala Val Phe Asn Tyr Gln Gly Leu 1 5 10 15 <210> 47 <211> 15 <212> PRT <213> Artificial <220> <223> Epitope <400> 47 Pro Val Thr Ala Val Phe Asn Tyr Gln Gly Leu Trp Arg Ser Cys 1 5 10 15 <210> 48 <211> 15 <212> PRT <213> Artificial <220> <223> Epitope <400> 48 Val Phe Asn Tyr Gln Gly Leu Trp Arg Ser Cys Val Arg Glu Ser 1 5 10 15 <210> 49 <211> 15 <212> PRT <213> Artificial <220> <223> Epitope <400> 49 Gln Gly Leu Trp Arg Ser Cys Val Arg Glu Ser Ser Gly Phe Thr 1 5 10 15 <210> 50 <211> 15 <212> PRT <213> Artificial <220> <223> Epitope <400> 50Arg Ser Cys Val Arg Glu Ser Ser Gly Phe Thr Glu Cys Arg Gly 1 5 10 15

Claims (38)

A pharmaceutical composition comprising an antibody for use in a method of treating or preventing a cancer disease, the method comprising administering the pharmaceutical composition to a patient in combination with an agent,
(a) the antibody binds to CLDN18.2 and mediates killing of cells expressing CLDN18.2 by complement-dependent cytotoxicity (CDC)-mediated lysis or antibody-dependent cytotoxicity (ADCC)-mediated lysis,
(b) The agent is an agent that stabilizes or increases the expression of CLDN18.2, and the agent is (i) oxaliplatin, (ii) 5-fluorouracil or capecitabine, (iii) 5-fluorouracil, leuco borine, and irinotecan, (iv) cisplatin and 5-fluorouracil or capecitabine, (v) 5-fluorouracil, folinic acid (leucovorin), and oxaliplatin; (vi) epirubicin, oxaliplatin and capecitabine; (vii) docetaxel, oxaliplatin, 5-fluorouracil and folinic acid; (viii) capecitabine and oxaliplatin; (ix) irinotecan; A pharmaceutical composition selected from the group consisting of (x) docetaxel and (xi) cisplatin. The method of claim 1, wherein the antibody heavy chain sequence comprises CDR1, CDR2 and CDR3 of the amino acid sequence shown in SEQ ID NO: 17, and the antibody light chain sequence comprises CDR1, CDR2 and CDR3 of the amino acid sequence shown in SEQ ID NO: 24. A pharmaceutical composition comprising a. The pharmaceutical composition according to claim 1, wherein the method further comprises administering an agent that stimulates γδ T cells. The pharmaceutical composition according to claim 3, wherein the γδ T cells are Vγ9Vδ2 T cells. The pharmaceutical composition according to claim 3, wherein the agent that stimulates γδ T cells is a bisphosphonate. The pharmaceutical composition according to claim 3, wherein the agent that stimulates γδ T cells is a nitrogen-containing bisphosphonate. The method of claim 3, wherein the agent that stimulates γδ T cells is zoledronic acid, clodronic acid, ibandronic acid, pamidronic acid, risedronic acid, minodronic acid, olpadronic acid, alendronic acid, incadronic acid and the like. A pharmaceutical composition selected from the group consisting of salts. The pharmaceutical composition according to any one of claims 3 to 7, wherein the agent that stimulates γδ T cells is administered in combination with interleukin-2. The pharmaceutical composition according to claim 1, wherein the antibody capable of binding to CLDN18.2 binds to the first extracellular loop of CLDN18.2. The method of claim 1, wherein the antibody having the ability to bind to CLDN18.2 is (i) accession numbers DSM ACC2737, DSM ACC2738, DSM ACC2739, DSM ACC2740, DSM ACC2741, DSM ACC2742, DSM ACC2743, DSM ACC2745, DSM ACC2746, DSM an antibody produced or obtainable from a clone deposited as ACC2747, DSM ACC2748, DSM ACC2808, DSM ACC2809, or DSM ACC2810, (ii) an antibody that is a chimeric or humanized form of the antibody of (i) above, (iii) an antibody of (i) above an antibody having the specificity of the antibody and (iv) an antibody containing the antigen-binding portion or antigen-binding site of the antibody of (i),
wherein said agent (b) is (i) oxaliplatin, (ii) 5-fluorouracil or capecitabine, (iii) 5-fluorouracil, leucovorin, and irinotecan, (iv) cisplatin and 5-fluorouracil. or capecitabine, (vii) docetaxel, oxaliplatin, 5-fluorouracil, and folinic acid. The pharmaceutical composition according to claim 1, wherein the method comprises administering an antibody capable of binding to CLDN18.2 at a dosage of up to 1000 mg/m ² . The pharmaceutical composition according to claim 1, wherein the method includes repeatedly administering an antibody capable of binding to CLDN18.2 at a dosage of 300 to 600 mg/m ² . The pharmaceutical composition according to claim 1, wherein the cancer is CLDN18.2 positive. The pharmaceutical composition according to claim 1, wherein the cancer is adenocarcinoma. The pharmaceutical composition according to claim 14, wherein the cancer is advanced adenocarcinoma. The pharmaceutical composition according to claim 1, wherein the cancer is selected from the group consisting of stomach cancer, esophagus cancer, gastroesophageal junction cancer, and gastroesophageal cancer. The pharmaceutical composition according to claim 16, wherein the cancer of the esophagus is a cancer of the lower esophagus. The pharmaceutical composition according to claim 1, wherein the patient is a HER2/neu negative patient or a patient with HER2/neu positive status but not treated with trastuzumab therapy. The pharmaceutical composition according to claim 1, wherein CLDN18.2 has an amino acid sequence according to SEQ ID NO: 1. A pharmaceutical composition comprising an agent for use in a method of treating or preventing a cancer disease, the method comprising administering the pharmaceutical composition to a patient in combination with an antibody,
(a) the antibody binds to CLDN18.2 and mediates killing of cells expressing CLDN18.2 by complement-dependent cytotoxicity (CDC)-mediated lysis or antibody-dependent cytotoxicity (ADCC)-mediated lysis,
(b) The agent is an agent that stabilizes or increases the expression of CLDN18.2, and the agent is (i) oxaliplatin, (ii) 5-fluorouracil or capecitabine, (iii) 5-fluorouracil, leuco borine, and irinotecan, (iv) cisplatin and 5-fluorouracil or capecitabine, (v) 5-fluorouracil, folinic acid (leucovorin), and oxaliplatin; (vi) epirubicin, oxaliplatin and capecitabine; (vii) docetaxel, oxaliplatin, 5-fluorouracil and folinic acid; (viii) capecitabine and oxaliplatin; (ix) irinotecan; A pharmaceutical composition for use in a method of treating or preventing a cancer disease, wherein the composition is selected from the group consisting of (x) docetaxel and (xi) cisplatin. 21. The method of claim 20, wherein the antibody heavy chain sequence comprises CDR1, CDR2 and CDR3 of the amino acid sequence shown in SEQ ID NO: 17, and the antibody light chain sequence comprises CDR1, CDR2 and CDR3 of the amino acid sequence shown in SEQ ID NO: 24. A pharmaceutical composition comprising a. The pharmaceutical composition according to claim 20, wherein the method further comprises administering an agent that stimulates γδ T cells. The pharmaceutical composition according to claim 22, wherein the γδ T cells are Vγ9Vδ2 T cells. The pharmaceutical composition according to claim 22, wherein the agent that stimulates γδ T cells is a bisphosphonate. 23. The pharmaceutical composition according to claim 22, wherein the agent that stimulates γδ T cells is a nitrogen-containing bisphosphonate. The method of claim 22, wherein the agent that stimulates γδ T cells is zoledronic acid, clodronic acid, ibandronic acid, pamidronic acid, risedronic acid, minodronic acid, olpadronic acid, alendronic acid, incadronic acid and the like. A pharmaceutical composition selected from the group consisting of salts. The pharmaceutical composition according to any one of claims 20 to 26, wherein the γδ T cells are administered in combination with interleukin-2. The pharmaceutical composition according to claim 20, wherein the antibody capable of binding to CLDN18.2 binds to the first extracellular loop of CLDN18.2. The method of claim 20, wherein the antibody capable of binding to CLDN18.2 is (i) accession numbers DSM ACC2737, DSM ACC2738, DSM ACC2739, DSM ACC2740, DSM ACC2741, DSM ACC2742, DSM ACC2743, DSM ACC2745, DSM ACC2746, DSM an antibody produced or obtainable from a clone deposited as ACC2747, DSM ACC2748, DSM ACC2808, DSM ACC2809, or DSM ACC2810, (ii) an antibody that is a chimeric or humanized form of the antibody of (i) above, (iii) an antibody of (i) above A pharmaceutical composition, wherein the pharmaceutical composition is an antibody selected from the group consisting of an antibody selected from the group consisting of an antibody having the specificity of the antibody and (iv) an antibody containing the antigen-binding portion or antigen-binding site of the antibody of (i). 21. The pharmaceutical composition according to claim 20, wherein the method comprises administering an antibody capable of binding to CLDN18.2 at a dosage of up to 1000 mg/m ² . The pharmaceutical composition according to claim 20, wherein the method includes repeatedly administering an antibody having the ability to bind to CLDN18.2 at a dosage of 300 to 600 mg/m ² . The pharmaceutical composition according to claim 20, wherein the cancer is CLDN18.2 positive. The pharmaceutical composition according to claim 20, wherein the cancer is adenocarcinoma. The pharmaceutical composition according to claim 33, wherein the cancer is advanced adenocarcinoma. The pharmaceutical composition according to claim 20, wherein the cancer is selected from the group consisting of stomach cancer, esophagus cancer, gastroesophageal junction cancer, and gastroesophageal cancer. The pharmaceutical composition according to claim 35, wherein the cancer of the esophagus is a cancer of the lower esophagus. The pharmaceutical composition according to claim 20, wherein the patient is a HER2/neu negative patient or a patient with HER2/neu positive status but not treated with trastuzumab therapy. 21. The pharmaceutical composition according to claim 20, wherein CLDN18.2 has an amino acid sequence according to SEQ ID NO: 1.